Terminal modifications of tumor necrosis factor

ABSTRACT

This invention relates to a novel physiologically active polypeptide containing modified amino acids on both terminals at the amino-terminus and the carboxy-terminus in the amino acid sequence of the human tumor neclosis factor (human TNF), a recombinant plasmid containing a DNA region encoding the polypeptide, a recombinant microorganism cell transformed with the recombinant plasmid, a method of producing the polypeptide by cultivating the microorganism cell, a pharmaceutical composition containing the polypeptide and a method of purifying and recovering the polypeptide. The polypeptide in this invention has excellent antitumor activity compared to the human TNF and can be utilized as an active ingredient of a pharmaceutical composition having antitumor activity.

TECHNOLOGICAL FIELD

This invention relates to a novel physiologically active polypeptide, arecombinant plasmid containing a DNA region encoding the polypeptide, arecombinant microorganism transformed with the plasmid, a method ofproducing the polypeptide using the microorganism cell, use of thepolypeptide and a method of recovering the purified polypeptide.

More specifically, it relates to a series of technologies in regard to anovel antitumorally active polypeptide.

In the present specification, an amino acid and a polypeptide will bedescribed abbreviatingly by the method accepted by Committee onBiochemical Nomenclature (CBN) of IUPAC-IUB, and for example, thefollowing abbreviations are used.

Ala: L-alanine

Arg: L-arginine

Asn: L-asparagine

Asp L-aspartic acid

Cys: L-cysteine

Gln: L-glutamine

Glu: L-glutamic acid

Gly: glycine

His: L-histidine

Ile: L-isoleucine

Leu: L-leucine

Lys: L-lysine

Met: L-methionine

Phe L-phenylalanine

Pro: L-proline

Ser: L-serine

Thr: L-threonine

Trp: L-tryptophan

Tyr: L-tyrosine

Val: L-valine

A DNA sequence will be expressed by the bases contained indeoxyribonucleotides constituting it, and for example, the followingabbreviations are used.

A: adenine (representing deoxyadenylic acid)

C: cytosine (representing deoxycytidylic acid)

G: guanine (representing deoxyguanylic acid)

T: thymine (representing deoxythymidylic acid)

(H₂ N)- and -(COOH) respectively show the aminoterminus andcarboxy-terminus of an amino acid sequence, 5 and (5')- and (3')-respectively show the 5'-terminus and the 3'-terminus of a DNA sequence.

TECHNOLOGICAL BACKGROUND

Carswell et al. found that a serum sample taken from a mouse stimulatedwith Bacillus Calmette-Guerin (BCG) and then given an endotoxin containsa substance which bleeds and necrotizes a solid tumor caused by atransplanted Meth A sarcoma; and named this substance tumor necrosisfactor (abbreviated as "TNF") (E. A. Carswell et al., Proc. Natl. Acad.Sci., U. S. A. 72, 3666 (1975)). TNF is found in many animals such asmice, rabbits and humans. Since it acts specifically on tumor cells ofany species, it is expected to be used as an antitumor agent.

Recently, Pennica et al. disclosed the primary structure of a human TNFprotein by cloning cDNA of human TNF, and reported on the expression ofthe human TNF gene in Escherichia coli (D. Pennica et al.: Nature, 312,724 (1984)). Later, Shirai et al. (T. Shirai et al.: Nature, 313, 803(1985)), Somura et al. [Somura et al.: Cancer and Chemotherapy, 12, 160(1985)), Wang et al. (A. M. Wang et al.: Science, 228, 149 (1985)), andMarmenout et al. (A. Marmenout et al.: Eur. J. Biochem., 152, 515(1985)) reported the expression of human TNF genes in E. coli.

Thus, large quantities of pure human TNF proteins have become availableby using the genetic manipulation technology, and physiologicalactivities of TNF other than the antitumor activity have been elucidatedin more detail. For example, it was suggested that cachectin, asubstance which is one cause of inducing cachexia in patients in theterminal stage of cancer or patients with serious infections, is verysimilar to TNF (B. Beulter et al.: "Nature", 316, 552 (1985)), and sincecachectin has lipoprotein lipase inhibitory activity, the administrationof TNF increases the amount of triglycerides in the blood, and maypossibly induce side-effects such as hyperlipemia. Elsewhere, theinfluence of TNF on vascular endothelial cells (J. R. Gamble et al.: J.Exp. Med., 162, 2163 (1985)), and its bone absorbing action (D. R.Beltolini et al.: "Nature", 319, 516 (1986)) have been reported.

On the other hand, the recent advance in genetic manipulation technologyhas enabled gene recombination to substitute an amino acid in a usefulprotein by another amino acid, to add an amino acid or to delete anamino acid from it. A number of research works have been conducted formodifying a naturally occurring protein and creating proteins which meeta specific purpose.

On the other hand, with regard to the modification of human TNFproteins, a number of research works have been made; there have beenreported substitution of either or both of amino acid residues of CYs⁶⁹and Cys¹⁰¹ by other amino acid residues in the amino acid sequence ofhuman TNF proteins shown in FIG. 1 ( see PCT Application Laid-OpenSpecification W086/04606 and Japanese Laid-Open Patent Publication No.263199/1987), substitution of Gly¹²² by another amino acid residue(Japanese Laid-Open Patent Publication Nos. 263199/1987 and 93799/1988),and substitution of Ala¹⁸ by another amino acid (Japanese Laid-openPatent Publication No. 87996/1988). Moreover, regarding the deletion ofamino acids on the amino-terminus side, it has been reported that TNF inwhich amino acids Nos. 1 to 6 are deleted has antitumor activity(Japanese Laid-Open Patent Publication No. 50923/1986), that TNF inwhich amino acids Nos. 1 to 7 are deleted has antitumor activity(Japanese Patent Application No. 90087/1986), that TNF in which aminoacids Nos. 1 to 10 are deleted has antitumor activity and the specificactivity is highest in TNF's in which animo acids Nos. 1 to 6, 1 to 7and 1 to 8 are deleted (PCT Application Laid-Open SpecificationW086/02381), that TNF in which amino acid Nos. 1 to 10 are deleted hasantitumor activity (Japanese Laid-Open Patent Publication No.272991/1987), that TNF in which amino acids Nos. 1 to 11 are deleted hascytotoxicity (Japanese Laid-Open Patent Publication No. 32486/1988), andthat regarding TNF in which amino acids Nos. 1 to 7 are deleted, TNFwith -Pro-Ser-Asp- amino acids Nos. 8 to 10 substituted by -Arg-Lys-Argamino acids are much increased in the specific activity (JapaneseLaid-Open Patent Publication No. 188396/1988).

Accordingly, the present inventors have made research works to producenovel modified human TNF proteins with a view to improving specificactivity and stability, broadening a zone of a reaction spectrum andreducing side effects, and consequently arrived at the presentinvention.

OBJECTS OF THE INVENTION

A first object of this invention is to provide a novel polypeptidehaving antitumor activity.

A second object of this invention is to provide a polypeptide havingincreased antitumor activity.

Another object of this invention is to provide a recombinant plasmidcontaining a DNA region encoding the novel polypeptide of thisinvention.

Still another object of this invention is to provide a recombinantmicroorganism cell transformed with the recombinant plasmid, and amethod of producing the polypeptide by cultivating the recombinantmicroorganism cell.

Yet another object of this invention is to provide a pharmaceuticalcomposition containing the polypeptide.

A further object of this invention is to provide a method of recoveringthe purified polypeptide.

The other objects of this invention will be clarified from the followingdescription.

DISCLOSURE OF THE INVENTION

According to the present inventors' research works, the objects andadvantages of this invention can be achieved by providing a novelphysiologically active polypeptide represented by formula (I) (SEQ IDNOS. 1-13 and 15-27)

    (NH.sub.2 -(Met).sub.n -A-X-B-COOH) . . . (I)

wherein

n is 0 or 1,

A denotes a bonding site, -Asp-, -Ser-Asp-, -Pro-Ser-Asp- or-Arg-Lys-Arg-,

B denotes -Ile-Ile-Ala-Phe-, -Ile-Ile-Ala-Trp-, -Ile-Ile-Ala-Leu-Phe-,-Ile-Ile-Trp-Leu-, Ile-Ile-Phe-Leu-, -Ile-Ile-Phe-Phe-,-Ile-Ile-Trp-Phe-, -Ile-Phe-Ala-Leu- or -Phe-Ile-Ala-Leu-, provided whenA is a bonding site, -Asp-, -Ser-Asp- or -Pro-Ser-Asp-, B is-Ile-Ile-Ala-Phe, and

X denotes-Lys-Pro-Val-Ala-His-Val-Val-Ala-Asn-Pro-Gln-Ala-Glu-Gly-Gln-Leu-Gln-Trp-Leu-Asn-Arg-Arg-Ala-Asn-Ala-Leu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-Asp-Asn-Gln-Leu-Val-Val-Pro-Ser-Glu-Gly-Leu-Tyr-Leu-Ile-Tyr-Ser-Gln-Val-Leu-Phe-Lys-Gly-Gln-Gly-Cys-Pro-Ser-Thr-His-Val-Leu-Leu-Thr-His-Thr-Ile-Ser-Arg-Ile-Ala-Val-Ser-Tyr-Gln-Thr-Lys-Val-Asn-Leu-Leu-Ser-Ala-Ile-Lys-Ser-Pro-Cys-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Tyr-Glu-Pro-Ile-Tyr-Leu-Gly-Gly-Val-Phe-Gln-Leu-Glu-Lys-Gly-Asp-Arg-Leu-Ser-Ala-Glu-Ile-Asn-Arg-Pro-Asp-Tyr-Leu-Asp-Phe-Ala-Glu-Ser-Gly-Gln-Val-Tyr-Phe-Gly-.

and a recombinant plasmid containing a DNA region encoding thepolypeptide, as well as a recombinant microorganism cell transformedwith the thus obtained recombinant plasmid, a method of producing thepolypeptide of formula (I) by cultivating the microorganism cell, apharmaceutical composition containing the polypeptide and a method ofpurifying and recovering the aforesaid polypeptide.

This invention will be described in more detail below.

The novel polypeptide represented by formula (I)

    (NH.sub.2)-(Met).sub.n -A-X-B-(COOH)                       (I)

wherein n, A, X and B are as defined above, in this invention ischaracterized in that both the amino-terminus and the carboxy-terminusin human TNF having a sequence of 157 amino acids Nos. 1 to 157 as shownin FIG. 1 are modified.

This point will be more specifically described. In formula (I), the unit-X- agrees with the amino acid sequence from No. 11 Lys to No. 153 Glyin the amino acid sequence of human TNF in FIG. 1. However, saidpolypeptide features that the (Met)_(n) -A- unit is bound to theamino-terminus and the -B- unit to the carboxy-terminus in the aminoacid sequence of this -X- and the amino acid sequences of both theterminus are not the same as those of both the terminus in the aminoacid sequence of human TNF in FIG. 1.

Thus, the polypeptide of formula (I) in this invention specificallycontains the following amino acid sequences (i) to (xiii) wherein n andx are as defined in formula [I].

    __________________________________________________________________________    (i)--(Met).sub.n --X--Ile--Ile--Ala--Phe--(SEQ ID NOS. 1 and 15)              (ii)--Met).sub.n --Asp--X--Ile--Ile--Ala--Phe--(SEQ ID NOS. 2 and 16)         (iii)--(Met).sub.n --Ser--Asp--X--Ile--Ile--Ala--Phe--(SEQ ID NOS. 3 and      17)                                                                           (iv)--(Met).sub.n --Pro--Ser--Asp--X--Ile--Ile--Ala--Phe--(SEQ ID NOS. 4      and 18)                                                                       (v)--(Met).sub.n --Arg--Lys--Arg--X--Ile--Ile--Ala--Phe--(SEQ ID NOS. 5       and 19)                                                                       (vi)--(Met).sub.n --Arg--Lys--Arg--X--Ile--Ile--Ala--Trp--(SEQ ID NOS. 6      and 20)                                                                       (vii)--(Met).sub.n --Arg--Lys--Arg--X--Ile--Ile--Ala--Leu--Phe--(SEQ ID       NOS. 7 and 21)                                                                (viii)--(Met).sub.n --Arg--Lys--Arg--X--Ile--Ile--Trp--Leu--(SEQ ID NOS.      8 and 22)                                                                     (ix)--(Met).sub.n --Arg--Lys--Arg--X--Ile--Ile--Phe--Leu--(SEQ ID NOS. 9      and 23)                                                                       (x)--(Met).sub.n --Arg--Lys--Arg--X--Ile--Ile--Phe--Phe--(SEQ ID NOS. 10      and 24)                                                                       (xi)--(Met).sub.n --Arg--Lys--Arg--X--Ile--Ile--Trp--Phe--(SEQ ID NOS. 11     and 25)                                                                       (xii)--(Met).sub.n -- Arg--Lys--Arg--X--Ile--Phe--Ala--Leu--(SEQ ID NOS.      12 and 26)                                                                    (xiii)--(Met).sub.n --Arg--Lys--Arg--X--Phe--Ile--Ala--Leu--(SEQ ID NOS.      13 and 27)                                                                    __________________________________________________________________________

The amino acid sequences (i) to (xiii) are roughly divided into (1) agroup consisting of (v) to (xiii) in which the amino-terminus [(Met)_(n)-A-] is -(Met)_(n) -Arg-Lys-Arg- and (2) a group consisting of (i) to(iv) in which the amino-terminus is different from -(Met)_(n)-Arg-Lys-Arg- and the carboxy terminus [-B-] is -lle-lle-Ala-Phe-.

Of the polypeptides represented by formula [I] in this invention, apolypeptide comprising a group of (v) to (xiii) in which the amino acidsequence of the amino-terminus is -(Met)_(n) -Arg-Lys-Arg- is preferableto that comprising the other group. Moreover, a polypeptide of (v),(vi), (viii) or (ix) in which the amino acid sequence of theamino-terminus is -(Met)_(n) -Arg-Lys-Arg and the amino acid sequence ofthe carboxy-terminus is

-Ile-Ile-Ala-Phe-,

-Ile-Ile-Ala-Trp-,

-Ile-Ile-Trp-Leu- or

-Ile-Ile-Phe-Leu-

is most preferable because of its highest antitumor activity.

The polypeptide of this invention is, as shown above, represented byformula (I) wherein -X- is not modified in comparison to the amino acidsequence of from No. 11 Lys to No. 153 Gly in the amino acid sequence ofhuman TNF (see FIG. 1). In the polypeptide of this invention, however,the amino acid sequence of this -X- permits some modification (e.g.substitution, deletion or addition) unless impairing the objects of thisinvention. For example, a polypeptide in which No. 45 Asp is substituedby Asn and No. 47 Gly by Ser or No. 54 Gly by Asp in the amino acidsequence (iv) still has the same activity as a polypeptide withunsubstituted -X-.

Moreover, according to this invention, there is provided a plasmid thathas a DNA sequence encoding the polypeptide of formula [I].

The plasmid of this invention is such that the intended polypeptide offormula (I) is produced by a microorganism cell obtained by transformingit in a host. Said plasmid contains not only the DNA sequence encodingthe poly peptide of formula (I) but also varied signal sequences used toexpress performance of vectors and control sequences of transcriptionand translation (e.g. an initiation codon, a termination codon, aninitiator, a terminator, an enhancer and a promotor).

According to this invention, there is thus provided a plasmid containingdouble-stranded DNA comprising single-stranded DNA and supplementalsingle-stranded DNA as represented by formula (II):

    (5')-(Yc).sub.p -Ac-Xc-Bc-(Zc).sub.q -(3')                 (II)

In the DNA sequence of formula (II), -Ac-, -Xc- and -Bc- correspondrespectively to DNA sequence portions encoding the amino acid sequences-A-, -X- and -B- in the polypeptide of formula (I). Yc and Zc meansignal sequences and/or control sequences of transcription andtranslation.

Thus, p or q in formula [II] is an optimum number for one geneexpression with good efficiency and is independently selected from 0, 1,2 and 3. DNA sequences -Yc-, -Ac-, -Xc-, -Bc- and -Zc- are concretelyshown below. However, they are one examples, and the DNA sequencesintroduced into the plasmid of this invention are not limited thereto.

First of all, as -Ac- and -Bc- in formula (II) are encoded in the aminoacid sequences -A- and -B- in formula (I), the DNA sequences are shownto correspond to same.

    __________________________________________________________________________    --A--                 --Ac--                                                  __________________________________________________________________________    --(bonding site)      --                                                      --Asp--               --GAC--                                                 --Ser--Asp--          --AGTGAC--                                              --Pro--Ser--Asp--     --CCGAGTGAC--                                           --Arg--Lys--Arg--     --CGTAAGCGC--                                           __________________________________________________________________________    --B--          --Bc--                                                         __________________________________________________________________________    --Ile--Ile--Ala--Phe--                                                                       --ATTATTGCCTTC--(SEQ ID NO. 39)                                --Ile--Ile--Ala--Trp--                                                                       --ATTATTGCCTGG--(SEQ ID NO. 40)                                --Ile--Ile--Ala--Leu--Phe--                                                                  --ATTATTGCCCTGTTC--(SEQ ID NO. 41)                             --Ile--Ile--Trp--Leu--                                                                       --ATTATTTGGCTG--(SEQ ID NO. 42)                                --Ile--Ile--Phe--Leu--                                                                       --ATTATTTTCCTG--(SEQ ID NO. 43)                                --Ile--Ile--Phe--Phe--                                                                       --ATTATTTTCTTC--(SEQ ID NO. 44)                                --Ile--Ile--Trp--Phe--                                                                       --ATTATTTGGTTC--(SEQ ID NO. 45)                                --Ile--Phe--Ala--Leu--                                                                       --ATTTTCGCCCTG--(SEQ ID NO. 46)                                --Phe--Ile--Ala--Leu--                                                                       --TTCATTGCCCTG--(SEQ ID NO. 47)                                __________________________________________________________________________

The DNA sequence of -Xc- encodes the amino acid sequence of -X- informula (I) as shown below. ##STR1##

-Yc- and -Zc- in formula [II] can be the following DNA sequences.##STR2##

The unit -Ac-Xc-Bc- in formula (II) is at least a DNA sequence encodingthe polypeptide of formula (I) in this invention.

According to this invention, the DNA sequence of formula (II) isintroduced into a vector plasmid to obtain a recombinant plasmid.Examples of the vector plasmid used in that case are a vector forEscherichia coli, a vector for Bacillus subtilis, and a vector foryeast. Above all, the vector for Eschericia coli is in generaladvantageously utilized. Examples of the vectors available to producethe plasmid are shown below. The parenthesized descriptions of thefollowing vectors show depositories and numbers, makers or literature'snames.

Vectors

(i) Vectors for Escherichia coli pBR322(ATCC 31344), pBR329(ATCC 37264),pACYC184(ATCC 37033), pDR540(ATCC 37282), pMB9(ATCC 37019),pDR720(Pharmacia), pUC9(ATCC 37252), pUC19(ATCC 37254),pUC13(Pharmacia), pPL-lambda(Pharmacia), pKK223-3(Pharmacia),pYS31N(S.Nakamura et al., J. Biotechnol., 8, 14 (1988)), pAA41(T. Masegiet al., Argic. Biol. Chem., 52, 1609 (1988))

Of these, pYS31N or pAA41 is preferable.

(ii) Vectors for Bacillus subtilis

For example, pBS7(ATCC 37280), pC194(ATCC 37034), pE194(ATCC 37128).

(iii) Vectors for yeast

For example, YEp13(ATCC 31125), YCp19(ATCC 37364), YRp7(ATCC 37060),YIp32(ATCC 37052), YRp17(ATCC 37078).

As is clear from the following description and Examples, this inventionproduces 13 recombinant plasmids that contain DNA sequences encoding theconcrete amino acid sequences of the polypeptide of formula [I]. Arelationship between numbers of the resulting recombinant plasmids andnumbers of the amino acid sequences is shown below.

    ______________________________________                                        Amino acid sequence No.                                                                        Recombinant plasmid No.                                      ______________________________________                                        (i)              pTNF630                                                      (ii)             pTNF629                                                      (iii)            pTNF628                                                      (iv)             pTNF621                                                      (v)              pTNF616                                                      (vi)             pTNF617                                                      (vii)            pTNF620                                                      (viii)           pTNF618                                                      (ix)             pTNF619                                                      (x)              pTNF633                                                      (xi)             pTNF634                                                      (xii)            pTNF643                                                      (xiii)           pTNF642                                                      ______________________________________                                    

Among the recombinant plasmids of this invention, (v) to (viii) arepreferable, and (v) pTNF616, (vi) pTNF617, (viii) pTNF618 and pTNF619are especially preferable.

The microorganism cell transformed with the recombinant plasmid in thisinvention may be that which can produce the polypeptide of formula (I)by the DNA sequence of formula (II) introduced into the plasmid.Examples thereof are Escherichia coli, Bacillus subtilis and yeast. Ofthere, Escherichia coli is preferable. Concrete examples of Escherichiacoli include C600r-m-(ATCC 33525), HB101 (ATCC 33694), W3110 (ATCC27325), DH1(ATCC 33849), JA221 (ATCC 33875), JM101 (ATCC 33876), (ATCC31244), RR1 (ATCC 31343) and LE392 (ATCC 33573).

Further, according to this invention, there is provided a method ofrecovering a purified polypeptide from an aqueous solution containing apolypeptide represented by formula (III) (SEQ ID NOS. 5-14 and 19-28)

    (NH.sub.2)-(Met).sub.n -A'-X-B'-(COOH)                     (III)

wherein

n is 0 or 1,

A' denotes -Arg-Lys-Arg-,

B' denotes -Ile-Ile-Ala-Phe-, Ile-Ile-Ala-Trp-, -Ile-Ile-Ala-Leu-Phe-,-Ile-Ile-Trp-Leu-, -Ile-Ile-Phe-Leu-, -Ile-Ile-Phe-Phe-,-Ile-Ile-Trp-Phe-, -Ile-Phe-Ala-Leu-, -Phe-Ile-Ala-Leu or-Ile-Ile-Ala-Leu-, and

X denotes the same amino acid sequence as in formula (I).

That is, the purified polypeptide of formula (III) can be recovered byeasy, simple operation which comprises cultivating an aqueous solutioncontaining the polypeptide of formula (III), e.g. a culture supernatantor a microorganism cell producing this polypeptide, then subjecting itto cell sonication, and subjecting the resulting lysate to the steps of

(1) contacting it with a cation exchange resin and adsorbing thepolypeptide to the cation exchange resin,

(2) rinsing the cation exchange resin to which the polypeptide isadsorbed with a solvent containing a salt in such concentration that thepolypeptide is substantially not eluted, and

(3) then eluting the cation exchange resin to which the polypeptide isadsorbed with a solvent containing a salt in such concentration that thepolypeptide can be eluted.

It is thought that such purifying and recovering method of thisinvention can be achieved because the amino acid sequence of theamino-terminus is -Arg-Lys-Arg- as is the case with the polypeptide offormula (III). Even if the aqueous solution containing the polypeptideof human TNF shown in FIG. 1 attached hereto is subjected to thetreating steps (1) to (3), a purified polypeptide cannot be obtained.

To explain briefly, the method of purifying and recovering thepolypeptide in this invention comprises adsorbing the polypeptide offormula (III) to the cation exchange resin, rinsing it with a solventcontaining a salt in such relatively low concentration that the adsorbedpolypeptide is substantially not eluted and then eluting the adsorbedpolypeptide upon increasing the salt concentration.

That the cation exchange resin can be used as a cation exchange columnchromatography in the method of purifying and recovering the polypeptideis excellent in conducting purification on an industrial scale in thatit is easy to make the polypeptide pyrogen-free and adsorption capacityis high.

The aqueous solution containing the polypeptide of formula (III), whichis subjected to the purifying and recovering method in this invention isa lysate obtained by collecting recombinant microorganism cellsproducing the polypeptide by centrifugation, suspending them in asuitable buffer, rupturing the cells with an ultrasonic generator andconducting centrifugation, or a sample formed by solubilizing aninsoluble fraction obtained in the same manner via a suitable method. Oran aqueous solution formed by dialyzing a culture solution of arecombinant microorganism cell secreting and producing the polypeptideagainst a suitable buffer.

As the cation exchange resin used in the step (1), a strongly acidic orweakly acidic cation exchange resin can be taken. Concrete examplesthereof are sulfopropyl cephalose, carboxymethyl cephalose andphosphocephalose.

The cation exchange resin can be used in an ordinary particulate,fibrous or membraneous form. Actually it is preferably used as a cationexchange column chromatography.

The polypeptide is adsorbed on the surface of the cation exchange resinby contacting the solution containing the polypeptide with the cationexchange resin. Since impurities other than the final polypeptide areadsorbed or adhered to the resulting adsorbed cation exchange resin, itis rinsed with a solvent containing a salt in such concentration thatthe adsorbed final polypeptide is substantially not eluted.

As the salt-containing solvent used in the rinsing of step (2), a bufferwith a salt concentration adjusted by a salt such as sodium chloride isemployed. The buffer can be a phosphate or tris-hydrochloride bufferwith pH adjusted to the vicinity of neutrality. Concrete examplesthereof are a 20 mM phosphate buffer (pH 7.4) and a 20 mMtris-hydrochloride buffer (pH 7.4).

The salt concentration is preferably at least 0.1 M but less than 0.15M. When it is less than 0.1 M, it is difficult to completely rinseimpurities except the final polypeptide. When it exceeds 0.15 M, thereis a possibility of eluting the final polypepride.

After rinsing, the final polypeptide is eluted from the cation exchangeresin to which the final polypeptide is adsorbed using a solventcontaining a salt in such concentration as to elute the finalpolypeptide.

The salt-containing solvent used in the elution of step (3) is used inthe form of a buffer in which the salt concentration is more increasedthan in the rinsing solution by a salt such as sodium chloride. Suchbuffer can be a phosphate or tris-hydrochloride buffer with pH adjustedto the vicinity of neutrality. Preferably, a 20 mN phosphate buffer (pH7.4) and a 20 mN tris-hydrochloride buffer (pH 7.4) are taken.

The salt concentration of the salt-containing solution as an elutingsolution varies with the final polypeptide, but at least 0.15 M isacceptable, and 0.18 M to 0.5 M is preferable. With the saltconcentration of 0.18 M to 0.5 M, the final polypeptide is obtained athigh purity. However, when it is higher than 0.5 M, impurities tend tobe eluted though in small amounts.

Thus, according to the aforesaid purifying and recovering method, thepyrogen-free high-purity polypeptide can be afforded by simple means,and the resulting polypeptide is available as an active ingredient of anantitumor pharmaceutical composition.

Embodiments to work this invention will be described in more detailbelow.

(A) Cloning of human TNF gene

Human TNF gene can be obtained by selecting several codons specifyingthe amino acids (D. Pennica et al. cited above) constituting the humanTNF protein, and chemically synthesizing the human TNF protein. Indesigning the human TNF gene, it is desired to select codons most suitedfor a host cell used, and to provide sites of cleavage with suitablerestriction endonucleases so as to permit easy coloning and genemodification later. Preferably, a DNA region encoding the human TNFprotein has a translation initiation codon (ATG) with the reading framecoinciding with its upstream, and a translation termination codon (TGA,TAG or TAA) with the reading frame coinciding with its downstream.Preferably in order to increase the expression efficiency, two or moretranslation termination codons are linked in tandem. Furthermore, byusing cleavage sites of endonucleases acting on its upstream anddownstream sides, this human TNF gene can be cloned into a suitablevector. An example of the base sequence of this human TNF gene is shownin FIG. 1.

Desirably, the human TNF gene designed as above is produced by dividingit into a plurality of oligonucleotides as shown in FIG. 2 with respectto each of the upper and lower chains, chemically synthesizing theseoligonucleotides, and then linking them with each other. Synthesismethods for the individual oligonucleotides include, for example, thediester method (H. G. Khorana, "Some Recent Developments in Chemistry ofPhosphate Esters of Biological Interest", John Wiley and Sons, Inc., NewYork (1961)), the triester method (R. L. Letsinger et al., J. Am. Chem.Soc., 89, 4801 (1967)) and the phosphite method (M. D. Matteucci et al.,Tetrahedron Lett., 21, 7190 (1980)). Synthesis by the phosphite methodusing an entirely automated DNA synthesizing machine is preferred fromthe view point of the synthesizing time, the yield and the simplicity ofthe operation. The synthesized oligonucleotides may be purified by, forexample, gel filtration, ion exchange chromatography, gelelectrophoresis, and high-performance liquid chromatography on areverse-phase column.

The hydroxyl groups of the 5'-teminus of the synthesizedoligonucleotides are phosphorylated with T4-tripolynucleotide kinase,for example. Then, the oligonucleotides are annealed, and linked withT4-DNA ligase, for example. To synthesize the human TNF gene by linkingthe synthetic oligonucleotides, it is preferred to divide theoligonucleotides into some blocks, linking them in each block, clone thelinked oligonucleotide blocks into a vector such as pBR322, and thenlinking the DNA fragments in these blocks. pTNF1BR, pTNF2N and pTNF3 arepreferably used as plasmids containing the DNA fragment blocksconstituting the human TNF gene.

After the cloned DNA fragments in the blocks constituting the human TNFgene are linked, the ligated DNA fragments may be joined to thedownstream end of the promoter SD sequence to produce an expressiongene. Usable promoters are, for example, trp promoter, lac promoter, trppromoter, P_(L) promoter and lpp promoter. The trp promoter isespecially preferred. Preferred plasmids having trp promoter are pYS31Nand pAA41. To increase the efficiency of expression, a terminater whichfunctions efficiently in E. coli can be attached downstream of the humanTNF gene. Examples of the terminater are an lpp gene terminater and atrp gene terminater. The trp A terminater is especially preferred.Preferably, pAA41 is used as a plasmid having the trp A terminater. Bycloning this human TNF gene into a vector derived, for example, frompBR322, an expression plasmid can be prepared. Preferably, pTNF401NN andpTNF401A are used as a plasmid expressing the human TNF gene.

(B) Cloning of a novel polypeptide gene having antitumor activity

The resulting plasmid expressing the human TNF gene is digested with asuitable restriction endonuclease to remove a specific region of thehuman TNF gene and then by using synthetic oligonucleotides having thesuitable base sequence, the gene is repaired. This technique permitspreparation of an expression plasmid containing a DNA encoding apolypeptide resulting from replacing a particular amino acid in thehuman TNF protein by another amino acid, deleting it, or adding anotheramino acid. Preferred plasmids expressing the novel antitumorally activepolypeptide gene are the 13 types of plasmids described above.

(C) Determination of the expression and eva1uation of the activity

E. coli, B. subtilis and yeasts are, for example, used as microorganismhosts for expressing the human TNF gene and the intended polypeptidegenes. E. coli is especially preferred. The above plasmid expressing thehuman TNF gene and the plasmid expressing the novel antitumorally activepolypeptide can be introduced into microorganism hosts such as E. coliC600r-m- strain (ATCC 33525) by a known method (M. V. Norgard et al.,Gene, 3, 279 (1978)).

The resulting recombinant microorganism cells are cultivated by methodsknown per se. M9 medium containing glucose and casamino acids may, forexample, be used as the culture medium [see T. Maniatis, et al.,"Molecular Cloning", p.440, Cold Spring Harbor Laboratory, New York(1982)]. As required, ampicillin, for example, is desirably added. Thecultivation is carried out under conditions suitable for the recombinantmicroorganism, for example, with aeration and stirring at 37° C. for 2to 36 hours. At the start of, or during, the cultivation, a chemicalsuch as 3-beta-indoleacrylic acid may be added in order to cause thepromoter to function efficiently.

After the cultivation, the recombinant microorganism cells are harvestedby, for example, centrifugal separation, and suspended in, for example,a phosphate buffer, and subjected to, for example, sonication to rupturethe recombinant microorganism cells. Subsequent centrifugal separationgives a lysate of the recombinant microorganism cells. The protein inthe lysate is separated by electrophoresis using a polyacrylamide gelcontaining sodium lauryl sulfate (SDS for short), and the protein in thegel is stained by a suitable method. By comparing the electrophoreticpatterns using the lysate of the microorganism cells not containing theexpression plasmid as a control, the expression of the human TNF gene orthe novel antitumorally active polypeptide gene is determined.

The antitumor activities of the resulting human TNF protein and novelantitumorally active polypeptide are evaluated by, for example the invivo activity measuring method by which the effect of necrotizing Meth Asarcoma transplanted in a mouse is examined (Carswell et al., citedhereinabove), or the in vitro activity measuring method by whichcytotoxicity on mouse L cells is examined (Ruff, J., Immunol., 126, 235(1981)). The in vitro activity measuring method is especiallyadvantageous in the aspect of simplicity.

The separation and purification of the human TNF protein and the novelantitumorally active polypeptide from the E. coli lysates may be carriedout in accordance with usually known protein separation and purificationmethods. Affinity column chromatography using antibody to the human TNFprotein is available. An example is affinity column chromatography usinga mouse monoclonal antibody to the human TNF protein.

Since the pyrogen-free high-purity polypeptide of this invention caneasily be obtained also by the above purifying and recovering methodusing the cation exchange resin, the aforesaid purifying and recoveringmethod in this invention is desirable.

The in vitro antitumor activity (as cited earlier) can directly becalculated by using the human TNF protein and antitumorally activepolypeptide purified products obtained by the abovementioned methods.

Thus, in accordance with this invention, the antitumorally activepolypeptide different from the known human TNF protein can be obtained,and the pharmaceutical composition having excellent antitumor activitycan be provided using this antitumorally active polypeptide.

(D) Preparation of a pharmaceutical composition

The polypeptides obtained as above in this invention are much higher inantitumor activity than the human TNF, and some have low side effects,especially low toxicity; they are therefore used as an antitumor agent.The pharmaceutical composition for this may be one containing thepolypeptide of formula (I) in this invention as an active ingredient anda pharmaceutically acceptable carrier.

When the pharmaceutical composition is prepared, the polypeptide used asthe active ingredient can be modified too with a known polymer such aspolyethylene glycol (PEG), dextran or poly-DL-alanine in order to reduceantigenicity of the polypeptide or enhance physiological activitythereof.

The pharmaceutical composition takes a form of an injection compositionor a suppository. As the injection composition, an intravenous injectioncomposition is especially preferable.

The injection composition is a mixture of a pharmaceutically acceptableamount of the polypeptide of formula (I) and a pharmaceuticallyacceptable carrier and can also contain an excipient which is commonlyadded to the injection composition, such as amino acids, carbohydrates,cellulose derivatives, polyvinyl pyrrolidone and inorganic compounds.Examples of the amino acids are glycine, alginine, alanine and theirpharmaceutically acceptable salts. Examples of the carbohydrates aremanitol, inositol, xylitol, lactol and glucose. Examples of thecellulose derivatives are sodium carboxymethylcellulose and methylcellulose. An example of the polyvinyl pyrrolidone is polyvinylpyrrolidone having a molecular weight of 10,000 to 1,000,000.

Examples of the organic acids are ascorbic acid, citric acid and theirsalts. Examples of the inorganic compounds are sodium hydrogenphosphate,sodium hydrogencarbonate and sodium acetate.

Examples of a solution that dissolves these excipients are distilledwater, physiological saline and Ringer's solution for injection.

The injection solution may contain, as required, a stabilizer, a surfaceactive agent, an isotonating agent, a soothing agent, an antiseptic anda buffer. Concrete examples thereof are antioxidants such as sodiumpyrosulfite and -ascorbic acid, and chelating agents such as EDTA andthioglycol as the stabilizer; nonionic surface active agents such aspolysolbate and polyoxyethylene derivatives as the surface active agent;sodium chloride as the isotonating agent; benzyl alcohol, xylocaine andprocaine as the soothing agent; parabens, chlorobutanol, benzalconiumchloride and thimerosal as the antiseptic; and sodium salts of citricacid, nitric acid and phosphoric acid as the buffer.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1 shows the base sequence of a designed human TNF gene;

FIG. 2 shows the base sequences of chemically synthesizedoligonucleotides;

FIGS. 3, 4 and 5 respectively show methods of preparing plasmidspTNF1BR, pTNF2N and pTNF3 having part of human TNF gene;

FIG. 6 shows a method of preparing plasmid pTNF401NN capable ofexpressing the human TNF gene;

FIG. 7-B shows a method of preparing a plasmid pAA41 using theoligonucleotide of FIG. 7-A;

FIG. 8 shows a method of preparing a plasmid pTNF401A capable ofexpressing the human TNF gene;

FIG. 9A and 9B show a method of preparing a plasmid pTNF471 capable ofexpressing the antitumorally active polypeptide gene;

FIGS. 10A(1), 10-A(2), and 10-B show a method of preparing plasmidscapable of expressing the antitumorally active polypeptide genes,pTNF616, pTNF617, pTNF618, pTNF619, pTNF620, pTNF633, pTNF634, pTNF642and pTNF643;

FIG. 11 shows a method of preparing pTNF416A;

FIGS. 12A and 12B and 13 show a method of preparing pTNF621 to pTNF627;

FIGS. 14-A(1), 14-A(2), 14A-(3) and 14-B show a method of preparingpTNF628 to pTNF632;

FIGS. 15-A, 15-B, 16-A and 16-B show a method of preparing pTNF637;

FIGS. 17-A, 17-B, 18, 19-A and 19-B show a method of preparing pTNF641;

FIG. 20 shows the results of expression of pTNF616;

FIG. 21 shows the results of measuring the in vitro antitumor activityof the antitumorally active polypeptide encoded by pTNF616;

FIG. 22 shows the results of measuring the in vitro antitumor activityof the antitumorally active polypeptide encoded by pTNF617;

FIG. 23 shows the results of measuring the in vitro antitumor activityof the antitumorally active polypeptide encoded by pTNF618;

FIG. 24 shows the results of measuring the in vitro antitumor activityof the antitumorally active polypeptide encoded by pTNF619;

FIG. 25 shows the results of measuring the in vitro antitumor activityof the antitumorally active polypeptide encoded by pTNF620;

FIG. 26 shows the results of measuring the in vitro antitumor activityof the antitumorally active polypeptide encoded by pTNF633 and pTNF634;

FIG. 27 shows the results of measuring the in vitro antitumor activityof the antitumorally active polypeptide encoded by pTNF642;

FIG. 28 shows the results of measuring the in vitro antitumor activityof the antitumorally active polypeptide encoded by pTNF643;

FIG. 29 shows the results of measuring the in vitro antitumor activityof the antitumorally active polypeptide encoded by pTNF637;

FIG. 30 shows the results of measuring the in vitro antitumor activityof the antitumorally active polypeptide encoded by pTNF641;

FIG. 31 shows the in vitro antitumor activity of the purifiedpolypeptide obtained in Example 9.

The following examples illustrate the present invention in greaterdetail. It should be understood however that the invention is notlimited to these examples.

EXAMPLE 1 Designing of a human TNF gene

A human TNF gene having the base sequence shown in FIG. 1 was designed.The base sequence of the structural gene portion of the human TNFprecursor cDNA reported by Pennica et al. (D. Pennica et al. Nature,312, 724 (1984)) was used as a basis. A cleavage site by a suitablerestriction endonuclease was provided at a suitable position. Atranslation initiation codon (ATG) was attached to the 5'-side and twotranslation termination codons (TGA and TAA), to the 3'-side of thehuman TNF gene respectively. A cleavage site by restriction endonucleaseClaI was provided upstream of the 5'-side translation initiation codonto maintain a proper distance between the translation initiation codonand the SD sequence in a suitable condition to permit joining of apromoter. A site of cleavage with restriction endonuclease HindIII wasprovided downstream of the 3'-side termination codons to permit easyjoining of a vector plasmid.

EXAMPLE 2 Chemical synthesis of oligonucleotides

The human TNF gene designed in Example 1 was divided into 17oligonucleotides as shown in FIG. 2. These oligonucleotides weresynthesized by the phosphite method using an entirely automated DNAsynthesizer (Model 380A made by Applied Biosystems). The synthesizedoligonucleotides were purified in accordance with the Manual of AppliedBiosystems, Inc.

Specifically, an aqueous ammonia solution containing the syntheticoligonucleotides was maintained overnight at 55° C. to remove theprotective groups of the DNA bases, and by gel filtration using aSephadex G-50 fine gel (Pharmacia), high-molecular-weight syntheticoligonucleotide fractions were recovered. The oligonucleotide fractionswere electrophoresed on a polyacrylamide gel containing 7M urea (gelconcentration 20 %), and the electrophoretic patterns were observed bythe ultraviolet shadowing method. Bands having the desired size were cutout. The polyacrylamide gel fragments were crushed finely, and 2 to 5 mlof a dissolving buffer (500 mM NH₄ OAc - 1 mM EDTA--0.1% SDS (pH 7.5))was added. The mixture was shaken overnight at 37° C. The aqueous layercontaining the desired DNA was recovered by centrifugal separation.Finally, the solution containing the synthetic oligonucleotides wascharged onto a gel filtration column (Sephadex G-50) to give purifiedproducts of the synthetic oligonucleotides. As required, thepolyacrylamide gel electrophoresis was repeated to increase the purityof the synthetic oligonucleotides.

EXAMPLE 3 Cloning of chemically synthesized human TNF gene

Using the 17 synthetic oligonucleotides (TNF-1 to TNF-17) prepared inExample 2, the human TNF gene was divided into three blocks and cloned.

The 5'-terminus of 0.1 to 1.0 microgram of each of the syntheticoligonucleotides TNF-2 to TNF-6 was phosphorylated with 5 to 15 units ofT4-polynucleotide kinase (E. coli B type, produced by Takara Shuzo Co.,Ltd.). The phosphorylation reaction was carried out in 10 to 20microliters of an aqueous solution of 50 mM Tris-HCl (pH 9.5), 10 mMMgCl₂, 5 mM dithiothreitol and 10 mM ATP at 37° C. for 30 minutes. Afterthe reaction, all aqueous solutions of synthetic oligonucleotides weremixed, and extracted with phenol and ether to deactivate and removeT4-polynucleotide kinase. Newly, 0.1 to 1.0 microgram of syntheticoligonucleotides TNF-1 and TNF-7 were added to the syntheticoligonucleotide mixture obtained. The mixture was heated to 90° C. andthen gradually cooled to room temperature to perform annealing. Themixture was dried under reduced pressure and dissolved in 30 microlitersof an aqueous solution of 66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl₂, 10 mMdithiothreitol and 1 mM ATP, and 300 units of T4-DNA ligase (a productof Takara Shuzo) was added. The ligating reaction was carried out at 11°C. for 15 hours. After the reaction, the reaction mixture waselectrophoresed on a polyacrylamide gel (gel concentration 5%), and theelectrophoretic patterns were observed by the ethidium bromide stainingmethod. Bands having the desired size (about 220 bp) were cut out, andby the method of Example 2, DNA was recovered from the polyacrylamidegel.

In the meantime, 3 micrograms of plasmid pBR 322 (about 4.4 kbp) for E.coli was dissolved in 30 microliters of an aqueous solution containing10 mM Tris-HCl (pH 7.5), 60 mM NaCl and 7 mM MgCl₂. Ten units ofrestriction endonuclease ClaI (a product of New England Bio-Rad) wasadded, and the digestion reaction was carried out at 30° C. for 1 hour.After the digestion, the reaction mixture was extracted with phenol andthen ether, and precipitated from ethanol to recover DNA. The DNA wasdissolved in 30 microliters of an aqueous solution containing 50 mMTris-HCl (pH 7.4), 100 mM NaCl and 10 mM MgSO₄, 10 units of restrictionenzyme SalI (a product of Takara Shuzo) was added, and the digestionreaction was carried out at 37° C. for 1 hour. After the reaction, thereaction mixture was electrophoresed on an agarose gel (gelconcentration 0.8%), and the cleavage patterns were observed by theethidium bromide staining method. A band corresponding to a DNA portionhaving a size of 3.7 kbp and containing most of the plasmid pBR 322 wascut out, and the agarose gel slice was dissolved in 3 times its amount(vol/wet) of an 8M aqueous solution of NaClO₄. A DNA fragment(ClaI--SalI) having a size of about 3.7 kbp was recovered from theagarose gel by the glass filter method of Chen et al. (C. W. Chen et al.Anal, Biochem., 101, 3339 (1980)).

The terminals of the DNA fragment having a size of about 220 bp andcontaining part of the human TNF gene, which has been obtainedpreviously, was phosphorylated in accordance with the method describedhereinabove, and the product was mixed with an aqueous solution of DNAhaving a size of about 3.7 kbp and containing most of the plasmid pBR322. After precipitation from ethanol, the two DNA fragments wereligated by the method described above.

Transformation of E. coli C600r-m- strain was carried out by an improvedmethod of the ordinary CaCl₂ method (the method of M. V. Norgard etal.). Specifically, a culture medium in which E. coli C600r-m- strainhad been cultivated for 18 hours was inoculated in 5 ml of L medium (1%tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7.2), and grown until theturbidity at 600 nm (OD₆₀₀) of the culture liquid containing the cellsreached 0.3. The cells were washed twice in a cold magnesium buffer (0.1M NaCl, 5 mM MgCl₂, 5 mM Tris-HCl (pH 7.0, 0° C.)], re-suspended in 2 mlof a cold calcium buffer 100 mM CaCl₂, 250 mM KCl, 5 mM MgCl₂, 5 mMTris-HCl (pH 7.6, 0° C.)], and left to stand at 0° C. for 25 minutes.The cells were concentrated to 1/10 of this volume in the calciumbuffer, and mixed with the aqueous DNA solution after the ligation in aratio of 2:1 (vol:vol). The mixture was maintained at 0° C. for 60minutes, 1 ml of LBG medium (1% tryptone, 0.5% yeast extract, 1% NaCl,0.08% glucose, pH 7.2) was then added, and the resulting mixture wascultivated with shaking at 37° C. for 1 hour. The culture broth wasinoculated in selective media (L-basal plates containing 30micrograms/ml of ampicillin (Sigma)) at a rate of 100 microliters/plate.The plates were cultivated at 30° C. overnight to grow thetransformants. DNA was prepared from the resulting ampicillin-resistantcolonies by a known method. By agarose gel electrophoresis, theproduction of the desired plasmid pTNF1BR (about 4.0 kbp) wasdetermined. FIG. 3 shows the method of preparing the plasmid pTNF1BR.

By the same procedure as above, plasmid pTNF2N (about 3.1 kbp) wasprepared by using synthetic oligonucleotides TNF-8 to TNF-13, andplasmid pTNF3 (about 2.4 kbp) by using synthetic oligonucleotides TNF-14to TNF-17. FIGS. 4 and 5 show methods of preparing the plasmids pTNF2Nand pTNF3.

It was determined by the method of Maxam and Gilbert (A. M. Maxam etal.: Methods in Enzymol., 65, 499 (1980)) that the syntheticoligonucleotide portions of the plasmids pTNF1BR, pTNF2N and pTNF3containing part of the human TNF gene obtained as above had the basesequences exactly as designed.

EXAMPLE 4 Preparation of a plasmid capable of expressing the human TNFgene

Ten micrograms of the plasmid pTNF1BR obtained in Example 3 was digestedwith restriction endonucleases ClaI and SalI as in Example 3. Thedigestion product was electrophoresed on a polyacrylamide gel (gelconcentration 5%). Then, in accordance with the method of Example 2, aDNA fragment (ClaI - SalI) having a size of about 220 bp and containingpart of the human TNF gene was recovered from the polyacrylamide gel.

Then, 10 micrograms of the plasmid pTNF2 obtained in Example 3 wasdissolved in 10 microliters of an aqueous solution containing 10 mMTris-HCl (pH 7.5), 60 mM NaCl, and 7 mM MgCl₂, and 40 units ofrestriction endonuclease PvuII (a product of Takara Shuzo) was added.The digestion reaction was carried out at 37° C. for 1 hour. Then, inaccordance with the method of Example 3, digestion with restrictionendonuclease SalI and polyacrylamide gel electrophoresis (gelconcentration 5%) were carried out. Thereafter, in accordance with themethod of Example 2, a DNA fragment (SalI - PvuII) having a size ofabout 170 bp and containing part of the human TNF gene was recoveredfrom the polyacrylamide gel.

Ten micrograms of the plasmid pTNF3 obtained in Example 3 was dissolvedin 100 microliters of an aqueous solution containing 10 mM Tris-HCl (pH7.5), 60 mM NaCl, and 7 mM MgCl₂, and 40 units of restrictionendonuclease PvuII and 40 units of restriction endonuclease HindIII (aproduct of Takara Shuzo) were added, and the digestion reaction wascarried out at 37° C. for 1 hour. After polyacrylamide gelelectrophoresis (gel concentration 5%), a DNA fragment (PvuII - HindIII)having a size of about 110 bp and containing part of the human TNF genewas recovered from the polyacrylamide gel in accordance with the methodof Example 2.

On the other hand, 5 micrograms of the plasmid pYS31N (about 4.7 kbp)containing E. coli trp promoter was digested as above with restrictionendonucleases ClaI and HindIII. After agarose gel electrophoresis (gelconcentration 0.8%), a DNA fragment (ClaI - HindIII) having a size ofabout 4.7 kbp and containing most of the plasmid pYS31N was recoveredfrom the agarose gel.

The resulting three DNA fragments having a size of about 220 bp, about170 bp and about 110 bp and containing part of the human TNF gene whichwere obtained as above were mixed with the DNA fragment (about 4.7 kbp)containing most of the plasmid pYS31N. After precipitation with ethanol,the mixture was subjected to ligating reaction with T4-DNA ligase. Afterthe reaction, in accordance with the method of Example 3, the ligationproduct was introduced into E. coli C600r-m- strain, and from thetransformants, clones having the desired plasmid pTNF401NN (about 6.2kbp) capable of expressing the human TNF gene were selected. FIG. 6shows a method of preparing the plasmid pTNF401NN.

Five micrograms of the plasmid pYS31N was partially digested withrestriction endonuclease PvuII and then digested with restrictionendonuclease HindIII. The digestion product was electrophoresed on anagarose gel (gel concentration 0.8%), and in accordance with the methodof Example 3, a DNA fragment (PvuII (2) - HindIII) having a size ofabout 2.7 kbp and containing trp promoter was recovered from the agarosegel.

Oligonucleotides having the base sequence shown in FIG. 7-A weresynthesized and purified in accordance with the method of Example 2. Theterminal of 0.5 microgram of each of the resulting two syntheticoligonucleotides was phosphorylated in accordance with the method ofExample 3. After annealing, the synthetic oligonucleotides were mixedwith the DNA fragment (PvuII (2) - HindIII) having a size of about 2.7kbp obtained previously. After precipitation with ethanol, the mixturewas subjected to a ligation reaction with T4-DNA ligase. After thereaction, the ligation product was introduced into E. coli C600r-m-strain in accordance with the method of Example 3. Clones having thedesired plasmid pAA41 (about 2.7 kbp) were selected from thetransformants. This plasmid is a high copy high efficient expressionvector resulting from removing the copy number control region from theplasmid pYS31N and joining E. coli trp A terminater to the downstream ofthe cloning site existing downstream of trp promoter. The method of itspreparation is shown in FIG. 7-B.

Two micrograms of the plasmid pAA41 was digested with restrictionendonucleases ClaI and HindIII in the same way as above, and afteragarose gel electrophoresis (gel concentration 0.8%), a DNA fragment(ClaI - HindIII) having a size of about 2.7 kbp and containing most ofthe pAA41 was recovered from the agarose gel.

Furthermore, 5 micrograms of the plasmid pTNF401NN capable of expressingthe human TNF gene, which had been obtained as above, was digested withrestriction endonucleases ClaI and HindIII in the same way as above.After polyacrylamide gel electrophoresis (gel concentration 5%), a DNAfragment (ClaI - HindIII) having a size of about 490 bp and containingthe entire region of the human TNF gene was recovered from thepolyacrylamide gel in accordance with the method of Example 2.

The DNA fragment (about 2.7 kbp) containing most of the plasmid pAA41and the DNA fragment (about 490 bp) containing the entire region of thehuman TNF gene obtained above were mixed, and after precipitation withethanol, subjected to a ligating reaction with T4-DNA ligase inaccordance with the method of Example 3. After the reaction, theligation product was introduced into E. coli C600r-m- strain, cloneshaving the desired plasmid pTNF401A (about 3.2 kbp) were selected fromthe transformants, in accordance with the method of Example 3. Thisplasmid has the ability to express the human TNF gene with goodefficiency, and FIG. 8 shows a method of its preparation.

EXAMPLE 5 Preparation of a plasmid capable of expressing theantitumorally active polypeptide

Twenty micrograms of the plasmid pTNF401A capable of expressing thehuman TNF gene obtained in Example 4 was digested with restrictionendonucleases ClaI and HindIII in accordance with the method of Example4. The digestion product was subjected to polyacrylamide gelelectrophoresis (gel concentration 5%) and agarose gel electrophoresis(gel concentration 0.8%). The resulting two DNA fragments (about 490 bpand about 2.7 kbp; both ClaI - HindIII) were recovered from the gels.

The DNA fragment (about 490 bp) containing the entire region of thehuman TNF gene was dissolved in 50 microliters of an aqueous solutioncontaining 10 mM Tris-HCl (pH 7.4), 10 mM MgSO₄ and 1 mM dithiothreitol,and 10 units of restriction endonuclease HapII (a product of TakaraShuzo) was added. The digestion reaction was carried out at 37° C. for 1hour. After the reaction, the reaction mixture was electrophoresed on apolyacrylamide gel (gel concentration 5%), and in accordance with themethod of Example 2, a DNA fragment (HapII - HindIII) having a size ofabout 390 bp and containing most of the human TNF gene was recoveredfrom the polyacrylamide gel.

Oligonucleotides having the base sequences shown in FIG. 9 weresynthesized and purified in accordance with the method of Example 2. Theterminals of the resulting four synthetic oligonucleotides in an amountof 0.5 microgram each were phosphorylated by the method of of Example 3,and after annealing, they were ligated by using T4-DNA ligase.

After the reaction, the resulting doublestranded oligonucleotide wasmixed with the DNA fragment (ClaI - HindIII) having a size of about 2.7kbp and the DNA fragment (HapII - HindIII) having a size of about 390 bpwhich were obtained above, and after precipitation with ethanol, themixture was subjected to ligation with T4-DNA ligase in accordance withthe method of Example 3. After the reaction, the ligation product wasintroduced into E. coli C600r-m- strain in accordance with the method ofExample 3, and clones having the desired plasmid pTNF471 (about 3.2 kbp)were selected from the transformants. This plasmid is a plasmid encodingthe antitumorally active polypeptide having the following amino acidsequence

    [NH.sub.2 ]-Arg-Lys-Arg-X-Ile-Ile-Ala-Leu-[COOH](SEQ ID NO. 14)

wherein X is as defined above, or a plasmid with Met bound to theamino-terminus (SEQ ID NO. 28). The method of its preparation is shownin FIG. 9.

Meanwhile, 20 micrograms of the above obtained human TNF gene expressingplasmid pTNF471 was digested with restriction endonuclease HindIII inaccordance with the method of Example 4. Subsequently, the digestionproduct was subjected to a digestion reaction with restrictionendonuclease NcoI (a product of Takara Shuzo) in an aqueous solutioncontaining 50 mM Tris-HCl (pH 7.4), 100 mM NaCl and 10 mM MgSo₄ at 37°C. for 1 hour. After the reaction, the resulting digestion product wassubjected to agarose gel electrophoresis (gel concentration 0.7%) andpolyacrylamide gel electrophoresis (gel concentration 5%). According tothe method in Example 2, the DNA fragment (NcoI - HindIII) having a sizeof about 140 bp and containing part of the human TNF gene was recoveredfrom the polyacrylamide gel. According to the method in Example 3, about3.0 kbp of the DNA fragment (NcoI - HindIII) containing most of pTNF471was recovered from the agarose gel.

Moreover, the above obtained DNA fragment (NcoI - HindIII) having a sizeof about 140 bp was dissolved in 50 microliters of an aqueous solutioncontaining 10 mM of Tris-HCl (pH 7.4), 10 mM MgSO₄ and 1 mMdithiothreitol, and 10 units of restriction endonuclease AccI (a productof Takara Shuzo) was added. The digestion reaction was conducted at 37°C. for 1 hour. After the reaction, polyacrylamide gel electrophoresis(gel concentration 8%) was carried out. In accordance with the method inExample 2, a DNA fragment (NcoI - AccI) having a size of about 110 bpand containing part of the human TNF gene was recovered from thepolyacrylamide gel.

Oligonucleotide having the base sequence No. 616 shown in FIG. 10A wassynthesized and purified according to the method in Example 2. Theterminals of the resulting two synthetic oligonucleotides in an amountof 0.5 microgram each were phosphorylated by the method in Example 3,followed by annealing.

After the annealing, the resulting double-stranded fragment (NcoI -HindIII) was mixed with the DNA fragment (NcoI - HindIII) having a sizeof about 3.0 kbp and and DNA fragment (NcoI - AccI) having a size ofabout 110 bp and containing part of the human TNF gene, which wereobtained above. After the mixture was precipitated with ethanol, theligation reaction with T4-DNA ligase was run by the method of Example 3.After the reaction, the resulting product was introduced into E. coliC600r-m- strain in accordance with the method of Example 3, cloneshaving the desired plasmid pTNF616 (about 3.2 kbp) were selected fromthe transformants. This plasmid is a plasmid capable of expressing anantitumorally active polypeptide gene encoding an antitumorally activepolypeptide having the following amino acid sequence

    (H.sub.2 N)-Arg-Lys-Arg-X-Ile-Ile-Ala-Phe-(COOH SEQ ID NO. 5))

wherein X is as defined above, or a polypeptide with Met bound to theamino-terminus (SEQ ID NO. 5). A method of preparing same is shown inFIG. 10 B.

In the above method, oligonucleotides having the following basesequences shown in FIG. 10 A were used instead of the base sequence No.616 shown in FIG. 10 A. Plasmids of corresponding numbers were obtained:

    __________________________________________________________________________    Base sequence No.                                                                       Plasmid                                                                            Amino acid sequence of polypeptide encoded by                  __________________________________________________________________________                   plasmid                                                        No. 617   pTNF617                                                                            (H.sub.2 N)--(Met).sub.n --Arg--Lys--Arg--X--Ile--Ile--Ala-                   -Trp--(COOH)                                                                  (SEQ ID NOS. 6 and 20)                                         No. 618   pTNF618                                                                            (H.sub.2 N)--(Met).sub.n --Arg--Lys--Arg--X--Ile--Ile--Trp-                   -Leu--(COOH)                                                                  (SEQ ID NOS. 8 and 22)                                         No. 619   pTNF619                                                                            (H.sub.2 N)--(Met).sub.n --Arg--Lys--Arg--X--Ile--Ile--Phe-                   -Leu--(COOH)                                                                  (SEQ ID NOS. 9 and 23)                                         No. 620   pTNF620                                                                            (H.sub.2 N)--(Met).sub.n --Arg--Lys--Arg--X--Ile--Ile--Ala-                   -Leu--Phe--(COOH)                                                             (SEQ ID NOS. 7 and 21)                                         No. 633   pTNF633                                                                            (H.sub.2 N)--(Met).sub.n --Arg--Lys--Arg--X--Ile--Ile--Phe-                   -Phe--(COOH)                                                                  (SEQ ID NOS. 10 and 24)                                        No. 634   pTNF634                                                                            (H.sub.2 N)--(Met).sub.n --Arg--Lys--Arg--X--Ile--Ile--Trp-                   -Phe--(COOH)                                                                  (SEQ ID NOS. 11 and 25)                                        No. 642   pTNF642                                                                            (H.sub.2 N)--(Met).sub.n --Arg--Lys--Arg--X--Phe--Ile--Ala-                   -Leu--(COOH)                                                                  (SEQ ID NOS. 13 and 27)                                        No. 643   pTNF643                                                                            (H.sub.2 N)--(Met).sub.n --Arg--Lys--Arg--X--Ile--Phe--Ala-                   -Leu--(COOH)                                                                  (SEQ ID NOS. 12 and 26)                                        __________________________________________________________________________

wherein n and X are as defined above.

Preparation of pTNF621 to pTNF632

Twenty micrograms of the plasmid pTNF401A capable of expressing thehuman TNF gene obtained in Example 4 was digested with restrictionendonucleases ClaI and HindIII in accordance with the method of Example4. The digestion product was subjected to polyacrylamide gelelectrophoresis (gel concentration 5%) and agarose gel electrophoresis(gel concentration 0.8%). In accordance with the methods of Examples 2and 3, the resulting two DNA fragments (about 490 bp and about 2.7 kbp,ClaI - HindIII in both cases) were recovered from the gels.

The DNA fragment having a size of about 490 bp and containing the entireregion of the human TNF gene was dissolved in 50 microliters of anaqueous solution containing 10 mM of Tris-HCl (pH 7.5), 60 mM of NaCland 7 mM of MgCl₂, and 10 units of restriction endonuclease AvaI (aproduct of Takara Shuzo) was added. The digestion reaction was conductedat 37° C. for 1 hour. After the reaction, polyacrylamide gelelectrophoresis (gel concentration 5%) was carried out, and a DNAfragment (AvaI - HindIII) having a size of about 460 bp and containingmost of the human TNF gene was recovered from the polyacrylamide gel inaccordance with the method of Example 2.

In accordance with the method of Example 2, the double-strandedoligonucleotide having the base sequence shown in FIG. 11 wassynthesized by dividing it into the upper and lower strands, andpurified. The terminals of the resulting four synthetic oligonucleotidesin an amount of 0.5 microgram each were phosphorylated by the method ofExample 3, followed by annealing.

After the annealing, the double-stranded oligonucleotide was mixed withthe DNA fragment (ClaI - HindIII) having a size of about 2.7 kbp and theDNA fragment (AvaII - HindIII) having a size of about 460 bp andcontaining most of the human TNF gene, which were obtained above. Afterthe mixture was precipitated with ethanol, the ligation reaction withT4-DNA ligase was carried out in accordance with the method of Example3. After the reaction, the ligation product was introduced into E. coliC600r-m- strain in accordance with the method of Example 3. Cloneshaving the desired plasmid pTNF416A (about 3.2 kbp) were selected fromthe transformants. This plasmid is a plasmid capable of expressing theantitumorally active polypeptide gene encoding the antitumorally activepolypeptide with No. 7 amino acid in the amino-terminus of TNF deleted.A method of preparing same is shown in FIG. 11.

Meanwhile, 20 micrograms of the above obtained expression plasmid pTNF416A was digested with restriction endonuclease HindIII in accordancewith the method of Example 4. The digestion reaction with restrictionendonuclease NcoI (a product of Takara Shuzo) was run at 37° C. for 1hour in an aqueous solution of 50 mM Tris-HCl (pH 7.4), 100 mN NaCl, 10mM MgSO₄ After the reaction, agarose gel electrophoresis (gelconcentration 0.7%) and polyacrylamide gel electrophoresis (gelconcentration 5% were conducted. A DNA fragment (NcoI - AccI) having asize of about 140 bp and containing part of the human TNF gene wasrecovered from the polyacrylamide gel in accordance with the method ofExample 2, and a DNA fragment (NcoI - HindIII) having a size of about3.0 kbp and containing most of pTNF416A was recovered from the agarosegel in accordance with the method of Example 3.

Moreover, the above DNA fragment (NcoI - HindIII) having a size of about140 bp was dissolved in 50 microliters of an aqueous solution containing10 mM Tris-HCl (pH 7.4), 10 mM MgSO₄ and 1 mM dithiothreitol, and 10units of restriction endonuclease AccI (a product of Takara Shuzo) wasadded. The digestion reaction was run at 37° C. for 1 hour. After thereaction, polyacrylamide gel electrophoresis (gel concentration 8%) wascarried out. In accordance with the method of Example 2, a DNA fragment(NcoI - AccI) having a size of about 110 bp and containing part of thehuman TNF gene was recovered from the polyacrylamide gel.

In accordance with the method of Example 2, oleonucleotide having thebase sequence shown in FIG. 12 was synthesized and purified. Theterminals of the resulting two synthetic oleonucleotides in an amount of0.5 microgram was phosphorylated by the method of Example 3, andfollowed by annealing.

After the annealing, the resulting double-stranded oleonucleotide wasmixed with the DNA fragment (NcoI - HindIII) having a size of about 3.0kbp and the DNA fragment (NcoI - AccI) having a size of about 110 kbpand containing part of the human TNF gene which were obtained above.After precipitation with ethanol, the ligation reaction with T4-DNAligase was run in accordance with the method of Example 3. After thereaction, the ligation product was introduced into E. coliC600r-m-strain. Clones having the desired plasmid pTNF621 (about 3.2kbp) were selected from the transformants. The plasmid is a plasmidcapable of expressing an antitumorally active poly peptide gene encodingan antitumorally active polypeptide having the following amino acidsequence

    (H.sub.2 N)-Pro-Ser-Asp-X-Ile-Ile-Ala-Phe-(COOH) (SEQ ID NO. 4)

or a polypeptide with Met bound to the amino-terminus (SEQ ID NO. 18). Amethod of preparing same is shown in FIG. 12.

Using synthetic DNA's (Nos. 622 to 627) shown in FIG. 13 instead of thesynthetic DNA having the base sequence shown in FIG. 11, there wasprepared a plasmid containing a DNA region encoding diluted TNF havingsuch a sequence that amino acid residue No. 1, 2, 3, 4, 5 or 6 at theamino-terminus in the amino acid sequence from No. 1 Val to No. 157 Leushown in FIG. 1 was diluted. 0n the other hand, in accordance with themethod shown in FIG. 14, there was prepared a plasmid containing a DNAregion encoding diluted TNF having such a sequence that amino acidresidue No. 8, 9, 10, 11 or 12 in the aminoterminus in the amino acidsequence from No. 1 Val to No. 157 Leu was diluted. Using the thusobtained plasmid of the DNA sequence encoding diluted TNF instead of theplasmid pTNF416A as a starting material in FIG. 12, there could beobtained the plasmid pTNF622, pTNF623, pTNF624, pTNF625, pTNF626,pTNF627, pTNF628, pTNF629, pTNF630, pTNF631 or pTNF632 containing a DNAregion encoding a novel physiologically active polypeptide having such asequence that in the amino acid sequence of from No. 1 Val to No. 157Leu shown in FIG. 1, amino acid residue No. 1, 2, 3, 4, 5, 6, 8, 9, 10,11 or 12 at the amino-terminus was diluted and No. 157 Leu was replacedwith Phe, or a polypeptide with Met bound to the amino-terminus.

Preparation of pTNF637

In accordance with the method of Example 3, the above obtainedexpression plasmid pTNF416A was digested with restriction endonucleasesEcoRI and SalI. After the digestion product was subjected topolyacrylamide gel electrophoresis (gel concentration 5%) and agarosegel electrophoresis (gel concentration 0.8%), the resulting two DNAfragments (about 560 bp and about 2.6 kbp, SalI - EcoRI in both cases)were recovered from the gels in accordance with the methods of Examples2 and 3.

The DNA fragment having a size of about 560 bp and containing half of a5'-site of the antitumorally active polypeptide gene was dissolved in 50microliters of an aqueous solution containing 10 mM Tris-HCl (pH 7.4),10 mM MgSO₄ and 1 mM dithiothreitol, and 10 units of restrictionendonuclease KpnI (a product of Takara Shuzo) was added. The digestionreaction was run at 37° C. for 1 hour. After the reaction,polyacrylamide gel electrophoresis (gel concentration 5%) was conducted,and a DNA fragment (EcoRI - KpnI) having a size of about 500 bp andcontaining a portion of the 5'-site of the antitumorally activepolypeptide gene was recovered from the polyacrylamide gel in accordancewith the method of Example 2.

In accordance with the method of Example 2, the oligonucreotide havingthe base sequence shown in FIG. 15 was synthesized and purified. Theterminals of the resulting two synthetic oligonucreotides in an amountof 0.5 microgram each were phosphorylated by the method of Example 3.After annealing, the ligation reaction with T4-DNA ligase was carriedout.

After the reaction, the resulting doublestranded oligonucleotide wasmixed with the DNA fragment (SalI - EcoRI) having a size of about 2.6kbp and the DNA fragment (EcoRI - KpnI) having a size of about 500 bpand containing a portion of the 5'-site of the antitumorally activepolypeptide gene, which were obtained above. After precipitation withethanol, the ligation reaction with T4-DNA ligase was conducted inaccordance with the method of Example 3. After the reaction, theligation product was introduced into E. coli C600r-m- strain inaccordance with the method of Example 3. Clones having the intendedplasmid pTNF611 (about 3.2 kbp) were selected from the transformants.This plasmid is an expression plasmid encoding an antitumorally activepolypeptide having the following amino acid sequence

    (H.sub.2 N)-Pro-Ser-Asp-X.sub.54 -Ile-Ile-Ala-Leu-(COOH (SEQ ID NO. 20))

wherein X₅₄ is a sequence in which No. 54 (number of the amino acidsequence in FIG. 1) Gly in the above defined X is replaced with Asp,

or a polypeptide with Met bound to the amino-terminus (SEQ. ID NO. 30).A method of preparing same is shown in FIG. 15.

Twenty micrograms of the resulting expression plasmid pTNF611 wasdigested with restriction endonuclease HindIII in accordance with themethod of Example 4. The digestion reaction with restrictionendonuclease NcoI (a product of Takara Shuzo) was carried out at 37° C.for 1 hour in an aqueous solution containing 50 mM Tris-HCl (pH 7.4),100 mM NaCl and 10 mM of MgSO₄.After the reaction, agarose gelelectrophoresis (gel concentration 0.7%) and polyacrylamide gelelectrophoresis (gel concentration 5%) were carried out. A DNA fragment(NcoI - HindIII) having a size of about 140 bp and containing part ofthe human TNF gene was recovered from the polyacrylamide gel inaccordance with the method of Example 2, and a DNA fragment (NcoI -HindIII) having a size of about 3.0 kbp and containing most of pTNF611was recovered from the agarose gel in accordance with the method ofExample 3.

Besides, the DNA fragment (NcoI - HindIII) having the size of about 140bp was dissolved in 50 microliters of an aqueous solution containing 10mM Tris-HCl (pH 7.4), 10 mM MgSO₄ and 1 mM dithiothreitol, and 10 unitsof restriction endonuclease AccI (a product of Takara Shuzo) was added.The digestion reaction was run at 37° C. for 1 hour. After the reaction,polyacrylamide gel electrophoresis (gel concentration 8%) was carriedout. In accordance with the method of Example 2, a DNA fragment (NcoI -AccI) having a size of about 110 bp and containing part of the human TNFgene was recovered from the polyacrylamide gel.

In accordance with the method of Example 2, oligonucleotide having thebase sequence shown in FIG. 16 was synthesized and purified. Inaccordance with the method of Example 3, the terminals of the resultingtwo synthetic oligonucleotides in an amount of 0.5 microgram each werephospholylated, followed by annealing.

After the annealing, the resulting double-stranded oligonucleotide wasmixed with the DNA fragment (NcoI - HindIII) having a size of about 3.0kbp and the DNA fragment (NcoI - AccI) having a size of about 110 bp andcontaining part of the human TNF gene. After precipitation with ethanol,the ligation reaction with T4-DNA ligase was run in accordance with themethod of Example 3. After the reaction, the ligation product wasintroduced into E. coli C600r-m- strain in accordance with the method ofExample 3. Clones having the intended plasmid pTNF637 (about 3.2 kbp)were selected from the transformants. This plasmid is a plasmid capableof expressing an antitumorally active polypeptide gene encoding anantitumorally active polypeptide having the following amino acidsequence

    (H.sub.2 N)-Pro-Ser-Asp-X.sub.54 -Ile-Ile-Ala-Phe-(COOH) (SEQ ID NO. 31)

wherein X₅₄ is as defined above, or a polypeptide with Met bound to theamino-terminus (SEQ ID NO. 32). A method of preparing same is shown inFIG. 16.

Preparation of pTNF641

Twenty micrograms of the plasmid pTNF401A capable of expressing thehuman TNF gene, which was obtained in Example 4, was dissolved in 100microliters of an aqueous solution containing 10 mM Tris-HCl (pH 7.4),10 mM MgSO₄ and 1 mM dithiothreitol, and 40 units of restrictionendonuclease KpnI (a product of Takara Shuzo) was added. The digestionreaction was run at 37° C. for 1 hour. Further, digestion withrestriction endonuclease ClaI was carried out in accordance with themethod of Example 3. After polyacrylamide gel electrophoresis (gelconcentration 5%) and agarose gel electrophoresis (gel concentration0.8%), the resulting two DNA fragments (about 160 bp and about 3.0 kbp,ClaI - KpnI in both cases) were recovered from the gels in accordancewith the methods of Examples 2 and 3.

The DNA fragment having a size of about 160 bp and containing the firsthalf of the human TNF gene was dissolved in 50 microliters of an aqueoussolution containing 10 mM Tris-HCl (pH 7.4), 10 mM MgSO₄ and 1 mMdithiothreitol, and 10 units of restriction endonuclease HapII (aproduct of Takara Shuzo) was added. The digestion reaction was run at37° C. for 1 hour. After the reaction, polyacrylamide gelelectrophoresis (gel concentration 5%) was carried out. In accordancewith the method of Example 2, a DNA fragment (ClaI - HapII) having asize of about 100 bp and containing the first half of the human TNF genewas recovered from the polyacrylamide gel.

In accordance with the method of Example 2, oligonuclease having thebase sequence shown in FIG. 17 was synthesized and purified. Inaccordance with the method of Example 3, the terminals of the resultingsynthetic two oligonucleotides in an amount of 0.5 microgram each werephosphorylated, followed by annealing.

After the reaction, the resulting doublestranded oligonucleotide wasmixed with the DNA fragment (ClaI - KpnI) having a size of about 3.0 kbpand the DNA fragment (ClaI-HapII) having a size of about 100 bp andcontaining the first half of the human TNF gene, which were obtainedabove. After precipitation with ethanol, the ligation reaction withT4-DNA ligase was run in accordance with the method of Example 3. Afterthe reaction, the ligation product was introduced into E. coli C600r-m-strain in accordance with the method of Example 3. Clones having theintended plasmid pTNF489 (about 3.2 kpb) were selected from thetransformants. This plasmid is a plasmid capable of expressing anantitumorally active polypeptide encoding an antitumorally activepolypeptide having the following amino acid sequence

    (H.sub.2 N)-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp-X.sub.45,47 -Ile-Ile-Ala-Leu-(COOH) (SEQ ID NO. 33)

wherein X₄₅,47 is a sequence that in the X sequence shown in FIG. 1, No.45 Asp is replaced with Asn and No. 47 Gln with Ser respectively,

or a polypeptide with Met bound to the amino-terminus (SEQ ID NO. 34). Amethod of preparing same is shown in FIG. 17.

Subsequently, 5 micrograms of the resulting plasmid pTNF 489 wasdissolved in 50 microliters of an aqueous solution containing 10 mMTris-HCl (pH 7.5), 60 mM NaCl and 7 mM MgSO₄, and 20 units ofrestriction endonuclease ClaI and 20 units of restriction endonucleaseAvaI (a product of Takara Shuzo) were added. The digestion reaction wasrun at 37° C. for 1 hour. After agarose gel electrophoresis (gelconcentration 0.7 %), a DNA fragment (ClaI - AvaI) having a size ofabout 3.2 kbp and containing most of the plasmid pTNF488 was recoveredfrom the agarose gel in accordance with the method of Example 3.

Meanwhile, in accordance with the method of Example 2, oligonucleotidehaving the base sequence shown in FIG. 18 was synthesized and purified.The resulting four synthetic oligonucreotides in an amount of 0.5microgram each were mixed in a combination shown in FIG. 18. Afterannealing, the mixture was mixed with the DNA fragment (ClaI - AvaI)having a size of about 3.2 kbp, which was obtained above. Afterprecipitation with ethanol, the ligation reaction with T4-DNA ligase wasconducted in accordance with the method of Example 3. After thereaction, the ligation product was introduced into E. coli C600r-m-strain in accordance with the method of Example 3. Crones having theintended plasmids pTNF497A and pTNF497B (about 3.2 kbp in both cases)were selected from the transformants. These plasmids are plasmidscapable of expressing an antitumorally active polypeptide gene encodingan antitumorally active polypeptide having the following amino acidsequence

    (H.sub.2 N)-Pro-Ser-Asp-X.sub.45,47 -Ile-Ile-Ala-Leu-(COOH)(SEQ ID NO. 35)

wherein X₄₅,47 is as defined above, or a polypeptide with Met bound tothe amino-terminus (SEQ. ID NO. 36). A method of preparing same is shownin FIG. 18.

Subsequently, 20 micrograms of the the plasmid pTNF497B capable ofexpressing the human TNF gene was digested with restriction endonucleaseHindIII in accordance with the method of Example 4. The digestionreaction with restriction endonuclease NcoI (a product of Takara Shuzo)was run in an aqueous solution containing 50 mM Tris-HCl (pH 7.4), 100mM NaCl and 10 mM MgSO₄ at 37° C. for 1 hour. After the reaction,agarose gel electrophoresis (gel concentration 0.7 %) and polyacrylamidegel electrophoresis (gel concentration 5 %) were conducted. A DNAfragment (NcoI - HindIII) having a size of about 140 bp and containingpart of the human TNF gene was recovered from the polyacrylamide gel inaccordance with the method of Example 2, and a DNA fragment (NcoI -HindIII) having a size of about 3.0 kbp and containing most of pTNF497Bwas recovered from the agarose gel in accordance with the method ofExample 3.

In addition, the above DNA fragment (NcoI - HindIII) having a size ofabout 140 bp was dissolved in 50 microliters of an aqueous solutioncontaining 10 mM Tris-HCl (pH 7.4), 10 mM MgSO₄ and 1 mM dithiothreitol,and 10 units of restriction endonuclease AccI (a product of TakaraShuzo) was added. The digestion reaction was run at 37° C. for 1 hour.After the reaction, polyacrylamide gel electrophoresis (gelconcentration 8%) was conducted. A DNA fragment (NcoI - AccI) having asize of about 110 bp and containing part of the human TNF gene wasrecovered from the polyacrylamide gel in accordance with the method ofExample 2.

In accordance with the method of Example 2, oligonucleotide having thebase sequence shown in FIG. 19 was synthesized and purified. Theterminals of the resulting two synthetic oligonucleotides in an amountof 0.5 microgram each were phosphorylated in accordance with the methodof Example 3, followed by annealing.

After the annealing, the resulting doublestranded oligonucleotide wasmixed with the DNA fragment (NcoI - HindIII) having a size of about 3.0kbp and the DNA fragment (NcoI - AccI) having a size of about 110 bp andcontaining part of the TNF fragment, which were obtained above. Afterprecipitation with ethanol, the ligation reaction with T4-DNA ligase wasconducted in accordance with the method of Example 3. After thereaction, the ligation product was introduced into E. coli C600r-m-strain in accordance with the method of Example 3. Clones having theintended plasmid pTNF641 (about 3.2 kbp) were selected from thetransformants. Said plasmid is a plasmid capable of expressing anantitumorally active polypeptide gene encoding an antitumorally activepolypeptide having the following amino acid sequence

    (H.sub.2 N)-Pro-Ser-Asp-X.sub.45,47 -Ile-Ile-Ala-Phe-(COOH)(SEQ ID NO. 37)

wherein X₄₅,47 is as defined above, or a polypeptide with Met bound tothe amino-terminus (SEQ ID NO. 38). A method of preparing same is shownin FIG. 19.

EXAMPLE 6 Determination of expression

E. coli C600r-m- strain having each of the human TNF gene expressingplasmid pTNF401A obtained in Example 4 and the expression plasmidpTNF471 or the expression plasmid pTNF616 obtained in Example 5, wasinoculated in 250 ml of M9 medium containing 30 to 50 micrograms/ml ofampicillin, 0.2% of glucose and 4 mg/ml of casamino acid an aqueoussolution (pH 7.4) of 0.6% NaHPO₄ -0.3% K₂ HPO₄ -0.05% NaCl-0.1% NH₄ Clwas sterilized in an autoclave, and an aqueous solution of MgSO₄ and anaqueous solution of CaCl₂, which had been separately sterilized in anautoclave, were added so that their final concentrations became 2 mM and0.1 mM respectively], and cultivated at 37° C. with shaking until OD₆₀₀reached 0.7 Then, 3-beta-indoleacrylic acid having a final concentrationof 50 micrograms/ml was added to the culture broth, and the cultivationwas continued further with shaking at 37° C. for 12 hours.

The E. coli cells were harvested by centrifugal separation, and washedwith a PBS buffer (20 mM phosphate buffer containing 150 mM NaCl, pH7.4). The washed cells were suspended in 10 ml of PBS buffer, andruptured by using an ultrasonic generator (Model 200M, Kubota), and thenthe solid residues were removed by centrifugal separation.

Tris-HCl buffer (pH 6.8) SDS, 2-mercaptoethanol and glycerol were addedto a portion of the resulting E. coli lysate so as to provide a finalconcentration of 60 mM, 2%, 4% and 10%, respectively, andSDS-polyacrylamide gel electrophoresis was performed (Suzuki, Iden(Genetics), 31, 43 (1977)). The concentration of the separating gel wasadjusted to 15%, and an SDS, Tris-glycine system (U. K. Laemmli, Nature,227, 680 (1970)) was used as an electrophoretic buffer. After theelectrophoresis, the proteins in the gel were stained with CoumassieBrilliant Blue R-250 (Bio-Rad), and the expression of the human TNF geneand the novel antitumorally active polypeptide gene was determined. Someof the results were copied and shown in FIG. 20.

The stained gel was subjected to a chromatoscanner (Model CS-930,Shimadzu), and the proportion of the produced antitumorally activepolypeptide in the E. coli cytoplasmic protein was calculated. It wasfound that in the E. coli having the human TNF gene expressing plasmidpTNF401A, about 19.4%, based on the total weight of the E. colicytoplasmic protein, of the antitumorally active polypeptide wasproduced, in E. coli having the expression plasmid pTNF471 about 20.3%,based on the total amount of the cytoplasmic protein, of theantitumorally active polypeptide was produced, and in E. coli having theexpression plasmid pTNF616, about 24.4% on the same basis, of theantitumorally active polypeptide was produced.

EXAMPLE 7 Evaluation of antitumor activity

The in vitro antitumor activity of the antitumorally active polypeptidewas measured in accordance with the method of Ruff et al. citedhereinabove.

Specifically, the E. coli lysate containing the antitumorally activepolypeptide obtained in Example 6 was diluted successively with amedium. The resulting sample (100 microliters) and 100 microliters of asuspension of mouse L-929 fibroblast cells (ATCC CCL-929) were mixed ina 96-well tissue-culture microtiter plate (Coaster). At this time,actinomycin D (Cosmegen, Banyu Pharmaceutical Co., Ltd.) was added to afinal concentration of 1 microgram/ml. Eagle's minimum essential medium(produced by Nissui Seiyaku) containing 5% (vol/vol) bovine fetal serumwas used as the medium. The microtiter plate was cultivated at 37° C.for 18 to 20 hours in air containing 5% carbon dioxide gas. Then, theliving cells were stained with a crystal violet solution (prepared bydissolving 0.5% (wt/vol) of crystal violet in a 5% (vol/vol) aqueoussolution of methanol). The excess of the crystal violet solution waswashed off, and the microtiter plate was dried. The remaining crystalviolet was extracted with 100 microliters of a 0.5% aqueous solution ofSDS, and the absorbance of the extract at 595 nm was measured by anELISA analyzer (model ETY-96, Toyo Sokki). This absorbance isproportional to the number of surviving cells. The dilution ratio of theE. coli lysate containing the antitumorally active polypeptide, whichcorresponds to 50% of the absorbance of the E. coli lysate to which nodiluting solution was added, was determined from a graph (for example,FIG. 21), and this dilution ratio is defined as one unit. It is clearfrom FIG. 21 that 100 microliters of the E. coli lysate containing thehuman TNF protein encoded by the expression plasmid pTNF401A has anactivity of about 7.6×10⁶ units; 100 microliters of the E. coli lysatecontaining the antitumorally active polypeptide encoded by theexpression plasmid pTNF471 has an activity of about 6.9×10⁷ units; and100 microliters of the E. coli lysate containing the antitumorallyactive polypeptide encoded by the expression plasmid pTNF616 has anactivity of about 1.9×10⁸ units.

The total amount of proteins contained in the E. coli lysates obtainedin Example 6 was determined by using a protein assay kit, and calculatedfrom a calibration curve prepared by using a bovine serum albumin. Fromthe amounts of expression, the activity values and the amounts ofproteins determined above, the specific activities of the antitumorallyactive polypeptide were calculated, and the results are shown inTable 1. Table 1 shows that the antitumorally active polypeptide encodedby pTNF616 has about 18 times as high a specific activity as the humanTNF protein, and about twice as high a specific activity as theantitumorally active polypeptide encoded by pTNF471.

                                      TABLE 1                                     __________________________________________________________________________    Comparison in antitumoractivity in vitro between human TNF protein            and antitumorally active polypeptide of this invention                                                           Antitumorally                                                                          Antitumorally                     Physiologically active polypeptide                                                                    Human TNF protein                                                                        active polypeptide                                                                     active polypeptide                __________________________________________________________________________    Plasmid                 pTNF 401A  pTNF 471 pTNF 616                           ##STR3##                19.4       20.3     24.4                             Activity (units/100 microliters-lyoate)                                                               7.6 × 10.sup.6                                                                     6.9 × 10.sup.7                                                                   1.9 × 10.sup.8              Total weight of E. coli cytoplasmic protein                                                           7.1        6.2      7.5                               (mg/ml-lysate)                                                                Specific activity       5.5 × 10.sup.7                                                                     5.5 × 10.sup.8                                                                   1.0 × 10.sup.9              (units/mg-physiologically active polypeptide)                                 __________________________________________________________________________

EXAMPLE 8

Regarding E. coli having each of the plasmids prepared in Example 5,expression was determined and antitumor activity in vitro was evaluatedin accordance with Examples 6 and 7. By the way, determination ofexpression and evaluation of antitumor activity were also conducted onE. coli having plasmid pTNF401 or pTNF471 for comparison as required.

Evaluation of the antitumorally active polypeptide encoded by pTNF617

The proportion of the antitumorally active polypeptide occupied in E.coli cytoplasmic protein of the antitumorally active polypeptide wascalculated according to Example 6. Consequently, it was found that in E.coli having the human gene expressing plasmid pTNF401A, 16.2%, based onthe total weight of the E. coli cytoplasmic protein, of the human TNFprotein was produced; in E. coli having the expression plasmid pTNF471,about 23.2%, on the same basis, of the antitumorally active polypeptidewas produced; and in E. coli having plasmid pTNF617 capable ofexpressing the antitumorally active polypeptide gene, about 18.7%, onthe same basis, of the antitumorally active polypeptide was produced.

In accordance with Example 7, a dilution ratio of E. coli lysatecorresponding to 50% of absorbance of E. coli lysate containing theantitumorally active polypeptide, etc., to which no diluting solutionwas added, was found by a graph (e.g. FIG. 22), and the very dilutionratio was defined as a unit. From FIG. 22, it became apparent that 100microliters of the E. coli lysate containing the human TNF proteinencoded by the expression plasmid pTNF401A has an activity of about 3.3×10⁶ units, 100 microliters of E. coli lysate containing theantitumorally active polypeptide encoded by the expression plasmidpTNF471 has an activity of about 3.7×10⁷ units, and 100 microliters ofthe E. coli lysate containing the antitumorally active polypeptideencoded by the expression plasmid pTNF617 has an activity of about4.0×10⁷ units.

The total amount of proteins contained in the E. coli lysates wasdetermined by using a protein assay kit (made by Bio-Rad), andcalculated from a calibration curve prepared by using a bovin serumalbumin. From the amounts of expression, the activity values and theamounts of proteins determined above, the specific activities of theantitumorally active polypeptide were calculated. The results are shownin Tables 4 and 5. From said Tables 4 and 5, it followed that theantitumorally active polypeptide encoded by pTNF617 has about 12 timesas high a specific activity as the human TNF protein, and about 1.4times as high a specific activity as the antitumorally activepolypeptide encoded by pTNF471.

Evaluation of the antitumorally active polypeptide encoded by pTNF618

The proportion of the antitumorally active polypeptide occupied in theE. coli cytoplasmic protein was calculated in accordance with Example 6.It was consequently found that in E. coli having the human TNF geneexpressing plasmid pTNF401A, 16.2%, based on the total weight of thecytoplasmic protein, of the human TNF gene was produced; in E. colihaving the expression plasmid pTNF471, about 23.2%, on the same basis,of the antitumorally active polypeptide was produced; and in E. colihaving the expression plasmid pTNF618, about 13.5%, on the same basis,of the antitumorally active polypeptide was produced.

In accordance with Example 7, a dilution ratio of the E. coli lysatecorresponding to 50% of absorbance of E. coli lysate containing theantitumorally active polypeptide, etc. was found by a graph (e.g. FIG.23), and the dilution ratio was defined as a unit. From FIG. 23, itbecame clear that 100 microliters of the E. coli lysate containing thehuman TNF protein encoded by the expression plasmid pTNF401A has anactivity of about 3.8×10⁶ units, 100 microliters of the E. coli lysatecontaining the antitumorally active polypeptide encoded by expressionplasmid pTNF471 has an activity of about 3.6×10⁷ units; and 100microliters of the E. coli lysate containing the antitumorally activepolypeptide encoded by the expression plasmid pTNF618 has an activity ofabout 3.9×10⁷ units.

The total amount of proteins contained in the E. coli lysates wasdetermined by using a protein assay kit (made by Bio-Rad), andcalculated from a calibration curve prepared by using a bovin serumalbumin. From the amounts of expression, the activity values and theamounts of proteins determined above, the specific activities of theantitumorally active polypeptide were calculated. The results are shownin Tables 4 and 5. Tables 4 and 5 revealed that the antitumorally activepolypeptide encoded by pTNF618 has about 14 times as high a specificactivity as the human TNF protein and about twice as high a specificactivity as the antitumorally active polypeptide encoded by pTNF471.

Evaluation of the antitumorally active polypeptide encoded by pTNF619

The proportion of the antitumorally active polypeptide occupied in theE. coli cytoplasmic protein was calculated in accordance with Example 6.As a result, it was found that in E. coli having the human TNF geneexpressing plasmid pTNF401A, about 16.3%, based on the total weight ofthe cytoplasmic protein, of the human TNF gene was produced; in E. colihaving the expression plasmid pTNF471, about 18.3%, based on the totalweight of the cytoplasmic protein, of the antitumorally activepolypeptide was produced; and in E. coli having the expression plasmidpTNF619, about 20.3%, on the same basis, of the antitumorally activepolypeptide was produced.

In accordance with Example 7, a dilution ratio of the E. coli lysatecorresponding to 50% of absorbance of E. coli lysate containing theantitumorally active polypeptide, etc., to which no diluting solutionwas added, was found by a graph (e.g. FIG. 24), and the dilution ratiowas defined as a unit. From FIG. 24, it was found that 100 microlitersof the E. coli lysate containing the human TNF protein encoded by theexpression plasmid pTNF401A has an activity of about 5.3×10⁶ units, 100microliters of the E. coli lysate containing the antitumorally activepolypeptide encoded by the expression plasmid pTNF471 has an activity ofabout 5.8×10⁷ units, and 100 microliters of the E. coli lysatecontaining the antitumorally active polypeptide encoded by theexpression plasmid pTNF619 has an activity of about 1.3×10⁸ units.

The total amount of the proteins contained in the E. coli lysates wasdetermined by using a protein assay kit (made by Bio-Rad), andcalculated from a calibration curve prepared by using a bovin serumalbumin. From the amounts of expression, the activity values and theamounts of proteins determined above, the specific activities of thelysates containing the antitumorally active polypeptides werecalculated. The results are shown in Tables 4 and 5. From Tables 4 and5, it became apparent that the antitumorally active polypeptide encodedby pTNF619 has about 18 times as high a specific activity as the humanTNF protein and about twice as high a specific activity as theantitumorally active polypeptide encoded by pTNF471.

Evaluation of the other antitumorally active polypeptides

One hundred microliters of the E. coli lysate containing theantitumorally active polypeptide encoded by the expression plasmidpTNF621 had an activity of about 10⁸ units, and was higher in activitythan the E. coli lysate containing the human TNF protein. One hundredmicroliters of the E. coli lysate containing the antitumorally activepolypeptide encoded by the expression plasmid pTNF637 had an activity ofabout 2×10³ units.

The total amount of the proteins contained in the E. coli lysate havingthe antitumorally active polypeptide encoded by the expression plasmidpTNF637 was determined by using a protein assay kit (made by BioRad),and calculated from a calibration curve prepared by using a bovin serumalbumin. From the amounts of expression, the activity values and theamounts of proteins determined above, the specific activity of thelysate containing the antitumorally active polypeptide was calculated,and found to be 8.3×10² (units/mg-protein).

Regarding pTNF's 620, 622, 624, 625, 626, 627, 628, 629, 630, 631, 632,633, 634, 642, 643 and 641, determination of expression and evaluationof antitumor activity in vivo were likewise conducted in accordance withExamples 6 and 7. FIGS. 25 to 30 show survival rate vs. lysate dilutionratio.

As a result, the activity per 100 microliters of the E. coli lysate isshown in Table 1, the ratio of the antitumorally active polypeptide inthe E. coli cytoplasmic protein in Table 2, the total concentration ofthe E. coli protein in the lysate in Table 3, the specific activity ofthe antitumorally active polypeptide in Table 4, and the specificactivity of the antitumorally active polypeptide given when the intactTNF (encoded by pTNF401A) is taken as 1 in Table 5, respectively. Itbecomes clear that the antitumorally active polypeptide produced from E.coli having the plasmid pTNF616, 617, 618, 619, 620, 643 or 641 ishigher in specific activity than the intact TNF.

EXAMPLE 9 Separation and purification of the antitumorally activepolypeptide

Separation and purification of the antitumorally active polypeptidesfrom the E. coli lysates having pTNF471, pTNF616 and pTNF619, which wereobtained in Example 5, were carried out by using carboxymethylsepharosecolumn chromatography (made by Pharmacia). First, the resin was packedin the column, and fully equilibrated with a 50 mN citrate buffer (pH6.2). The lysate containing the antitumorally active polypeptide,obtained in Example 2, was then charged onto said column. After theresulting substance was thoroughly washed with PBS buffer 20 mMphosphate buffer (pH 7.4) containing 140 mM NaCl, the antitumorallyactive polypeptide was eluted with 20 mM phosphate buffer (pH 7.4)containing 420 mM NaCl. The eluted antitumorally active polypeptide wassubjected to SDS-polyacrylamide gel electrophoresis (a concentration ofa separation gel 15%). After the electrophoresis, protein bands in thegel were stained with a dye. Consequently, one band was observed only ina position of a molecular weight of about 15,000 to 17,000. It wasconfirmed that the antitumorally active polypeptide having a purity of98% was obtained.

EXAMPLE 10 Evaluation of activity of the purified product

The antitumor activity in vitro of the antitumorally active polypeptidespurified in Example 9 was evaluated as in Example 7 according to theRuff's method. FIG. 31 revealed that 1 mg of the purified product of theantitumorally active polypeptide encoded by the expression plasmidpTNF471 has an activity of about 8.3×10⁸ units, 1 mg of the purifiedproduct of the antituplasmid pTNF616 has an activity of about 1.2×10⁹units, and 1 mg of the purified product of the antitumorally activepolypeptide encoded by the expression plasmid pTNF619 has an activity ofabout 1.6×10⁹ units.

                                      TABLE 2                                     __________________________________________________________________________    Activity per 100 microliters of E. coli lysate                                (10.sup.7 units/100 microliters of lysate)                                           Test No.                                                               Plasmid                                                                              1  2  3  4  5  6 7  8  9  10                                                                              11    12                                   __________________________________________________________________________    pTNF 401A                                                                            0.76                                                                             0.33                                                                             0.38                                                                             0.53                                                                             0.42       0.11       1.3                                  pTNF 471                                                                             6.9                                                                              3.7                                                                              3.6                                                                              5.8                                                           pTNF 616                                                                             19                                                                     pTNF 617  4.0                                                                 pTNF 618     3.9                                                              pTNF 619        13                                                            pTNF 620           4.6                                                        pTNF 633              9.6                                                     pTNF 634                0.59                                                  pTNF 642                   0.05                                               pTNF 643                      0.28                                            pTNF 621                         10                                           pTNF 637                           2 × 10.sup.-4                        pTNF 641                                 1.2                                  __________________________________________________________________________

                                      TABLE 3                                     __________________________________________________________________________    Proportion of the antitumorally active polypeptide occupied                   in the E. coli cytoplastic protein                                            (antitumorally active polypeptide/total weight of E. coli                     cytoplastic protein; unit %)                                                         Test No.                                                               Plasmid                                                                              1  2  3  4  5  6 7 8 9  10                                                                              11                                                                              12                                         __________________________________________________________________________    pTNF 401A                                                                            19.4                                                                             16.2                                                                             16.2                                                                             16.3                                                                             16.2     13.7   17                                         pTNF 471                                                                             20.3                                                                             23.2                                                                             23.2                                                                             18.3                                                          pTNF 616                                                                             24.4                                                                   pTNF 617  18.7                                                                pTNF 618     13.5                                                             pTNF 619        20.3                                                          pTNF 620           26.6                                                       pTNF 633              --                                                      pTNF 634                --                                                    pTNF 642                  --                                                  pTNF 643                    13.9                                              pTNF 621                       --                                             pTNF 637                         --                                           pTNF 641                           10                                         __________________________________________________________________________

                                      TABLE 4                                     __________________________________________________________________________    Total amount of E. coli proteins in lysates                                   (mg/ml-lysate)                                                                       Test No.                                                               Plasmid                                                                              1  2  3  4  5  6 7 8 9  10                                                                              11                                                                              12                                         __________________________________________________________________________    pTNF 401A                                                                            7.1                                                                              6.6                                                                              6.6                                                                              6.5         4.4    10.2                                       pTNF 471                                                                             6.2                                                                              6.2                                                                              6.2                                                                              7.1                                                           pTNF 616                                                                             7.5                                                                    pTNF 617  6.0                                                                 pTNF 618     5.9                                                              pTNF 619        7.2                                                           pTNF 620           6.5                                                        pTNF 633              --                                                      pTNF 634                --                                                    pTNF 642                  --                                                  pTNF 643                    3.7                                               pTNF 621                                                                      pTNF 637                                                                      pTNF 641                           8.9                                        __________________________________________________________________________

                                      TABLE 5                                     __________________________________________________________________________    Specific activity of the antitumorally active polypeptide                     (10.sup.8 units/mg-antitumorally active polypeptide)                                 Test No.                                                               Plasmid                                                                              1  2  3  4  5  6 7 8 9  10                                                                              11    12                                     __________________________________________________________________________    pTNF 401A                                                                            0.55                                                                             0.31                                                                             0.35                                                                             0.50                                                                             0.39     0.19       0.75                                   pTNF 471                                                                             5.5                                                                              2.6                                                                              2.5                                                                              4.4                                                           pTNF 616                                                                             10                                                                     pTNF 617  3.6                                                                 pTNF 618     4.8                                                              pTNF 619        8.9                                                           pTNF 620           2.7                                                        pTNF 633                                                                      pTNF 634                                                                      pTNF 642                                                                      pTNF 643                    0.54                                              pTNF 621                       --                                             pTNF 637                         8.3 × 10.sup.-6                        pTNF 641                               1.3                                    __________________________________________________________________________

                                      TABLE 6                                     __________________________________________________________________________    Specific activity of the antitumorally active polypeptide                     when intact polypeptide (pTNF401A) is taken as 1                                     Test No.                                                               Plasmid 1  2  3  4  5 6 7 8 9 10                                                                              11                                                                              12                                          __________________________________________________________________________    pTNF 401A                                                                             1  1  1  1  1 --                                                                              --                                                                              --                                                                              --                                                                              --                                                                              --                                                                              1                                           pTNF 471                                                                              10 8.4                                                                              7.1                                                                              8.8                                                          pTNF 616                                                                              18.2                                                                  pTNF 617   11.6                                                               pTNF 618      13.7                                                            pTNF 619         17.8                                                         pTNF 620            6.9                                                       pTNF 633              --                                                      pTNF 634                --                                                    pTNF 642                  --                                                  pTNF 643                    2.8                                               pTNF 621                      --                                              pTNF 637                        --                                            pTNF 641                          1.7                                         __________________________________________________________________________

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 50                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 147 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 ( B) LOCATION:                                                                (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       LysProValAlaHisValValAlaAsnProGlnAlaGluGlyGln                                 151015                                                                        LeuGlnTrpLeuAsnA rgArgAlaAsnAlaLeuLeuAlaAsnGly                                202530                                                                        ValGluLeuArgAspAsnGlnLeuValValProSerGluGlyLeu                                 35 4045                                                                       TyrLeuIleTyrSerGlnValLeuPheLysGlyGlnGlyCysPro                                 505560                                                                        SerThrHisValLeuLeuThrHisThrIleSerArgIle AlaVal                                657075                                                                        SerTyrGlnThrLysValAsnLeuLeuSerAlaIleLysSerPro                                 808590                                                                        CysG lnArgGluThrProGluGlyAlaGluAlaLysProTrpTyr                                95100105                                                                      GluProIleTyrLeuGlyGlyValPheGlnLeuGluLysGlyAsp                                 110 115120                                                                    ArgLeuSerAlaGluIleAsnArgProAspTyrLeuAspPheAla                                 125130135                                                                     GluSerGlyGlnValTyrPheGlyIle IleAlaPhe                                         140145                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 148 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                   (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AspLysProValAlaHisValValAlaAsnProGlnAlaGluGly                                 151015                                                                        GlnLeuGlnTrpLeuAsnArgArg AlaAsnAlaLeuLeuAlaAsn                                202530                                                                        GlyValGluLeuArgAspAsnGlnLeuValValProSerGluGly                                 3540 45                                                                       LeuTyrLeuIleTyrSerGlnValLeuPheLysGlyGlnGlyCys                                 505560                                                                        ProSerThrHisValLeuLeuThrHisThrIleSerArgIleAla                                  657075                                                                       ValSerTyrGlnThrLysValAsnLeuLeuSerAlaIleLysSer                                 808590                                                                        ProCysGlnArg GluThrProGluGlyAlaGluAlaLysProTrp                                95100105                                                                      TyrGluProIleTyrLeuGlyGlyValPheGlnLeuGluLysGly                                 110 115120                                                                    AspArgLeuSerAlaGluIleAsnArgProAspTyrLeuAspPhe                                 125130135                                                                     AlaGluSerGlyGlnValTyrPheGlyIleIleAla Phe                                      140145                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 149 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       SerAspLysProValAlaHisValValAlaAsnProGlnAlaGlu                                 151015                                                                        GlyGlnLeuGlnTrpLeuAsnArgArgAla AsnAlaLeuLeuAla                                202530                                                                        AsnGlyValGluLeuArgAspAsnGlnLeuValValProSerGlu                                 3540 45                                                                       GlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGlyGlnGly                                 505560                                                                        CysProSerThrHisValLeuLeuThrHisThrIleSerArgIle                                  657075                                                                       AlaValSerTyrGlnThrLysValAsnLeuLeuSerAlaIleLys                                 808590                                                                        SerProCysGlnArgGlu ThrProGluGlyAlaGluAlaLysPro                                95100105                                                                      TrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeuGluLys                                 110115 120                                                                    GlyAspArgLeuSerAlaGluIleAsnArgProAspTyrLeuAsp                                 125130135                                                                     PheAlaGluSerGlyGlnValTyrPheGlyIleIleAlaPh e                                   140145                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ProSerAspLysProValAlaHisValValAlaAsnProGlnAla                                 151015                                                                        GluGlyGlnLeuGlnTrpLeuAsnArgArgAl aAsnAlaLeuLeu                                202530                                                                        AlaAsnGlyValGluLeuArgAspAsnGlnLeuValValProSer                                 35404 5                                                                       GluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGlyGln                                 505560                                                                        GlyCysProSerThrHisValLeuLeuThrHisThrIleSerArg                                  657075                                                                       IleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAlaIle                                 808590                                                                        LysSerProCysGlnArgGl uThrProGluGlyAlaGluAlaLys                                95100105                                                                      ProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeuGlu                                 110115 120                                                                    LysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyrLeu                                 125130135                                                                     AspPheAlaGluSerGlyGlnValTyrPheGlyIleIleAlaP he                                140145150                                                                     (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 ( B) LOCATION:                                                                (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ArgLysArgLysProValAlaHisValValAlaAsnProGlnAla                                 151015                                                                        GluGlyGlnLeuGlnT rpLeuAsnArgArgAlaAsnAlaLeuLeu                                202530                                                                        AlaAsnGlyValGluLeuArgAspAsnGlnLeuValValProSer                                 35 4045                                                                       GluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGlyGln                                 505560                                                                        GlyCysProSerThrHisValLeuLeuThrHisThrIle SerArg                                657075                                                                        IleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAlaIle                                 808590                                                                        LysSe rProCysGlnArgGluThrProGluGlyAlaGluAlaLys                                95100105                                                                      ProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeuGlu                                 110 115120                                                                    LysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyrLeu                                 125130135                                                                     AspPheAlaGluSerGlyGlnValTyr PheGlyIleIleAlaPhe                                140145150                                                                     (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ArgLysArgLysProValAlaHisValValAlaAsnProGlnAla                                 151015                                                                         GluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeuLeu                                202530                                                                        AlaAsnGlyValGluLeuArgAspAsnGlnLeuValValProSer                                  354045                                                                       GluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGlyGln                                 505560                                                                        GlyCysProSerThrHisValLeu LeuThrHisThrIleSerArg                                657075                                                                        IleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAlaIle                                 8085 90                                                                       LysSerProCysGlnArgGluThrProGluGlyAlaGluAlaLys                                 95100105                                                                      ProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeuGlu                                  110115120                                                                    LysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyrLeu                                 125130135                                                                     AspPheAlaGlu SerGlyGlnValTyrPheGlyIleIleAlaTrp                                140145150                                                                     (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 151 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ArgLysArgLysProValAlaHisValValAlaAsnProGlnAla                                 1510 15                                                                       GluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeuLeu                                 202530                                                                        AlaAsnGlyValGluLeuArgAspAsnGlnLeuValValPro Ser                                354045                                                                        GluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGlyGln                                 505560                                                                        GlyCysP roSerThrHisValLeuLeuThrHisThrIleSerArg                                657075                                                                        IleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAlaIle                                 80 8590                                                                       LysSerProCysGlnArgGluThrProGluGlyAlaGluAlaLys                                 95100105                                                                      ProTrpTyrGluProIleTyrLeuGlyGly ValPheGlnLeuGlu                                110115120                                                                     LysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyrLeu                                 125130 135                                                                    AspPheAlaGluSerGlyGlnValTyrPheGlyIleIleAlaLeu                                 140145150                                                                     Phe                                                                           (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 amino acids                                                   (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ArgLysArgLysProValAlaHisValValAlaAsnProGlnAla                                 1 51015                                                                       GluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeuLeu                                 202530                                                                        AlaAsnGlyValGluLeuArgA spAsnGlnLeuValValProSer                                354045                                                                        GluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGlyGln                                 5055 60                                                                       GlyCysProSerThrHisValLeuLeuThrHisThrIleSerArg                                 657075                                                                        IleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAlaIle                                  808590                                                                       LysSerProCysGlnArgGluThrProGluGlyAlaGluAlaLys                                 95100105                                                                      ProTrpTyrG luProIleTyrLeuGlyGlyValPheGlnLeuGlu                                110115120                                                                     LysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyrLeu                                 125 130135                                                                    AspPheAlaGluSerGlyGlnValTyrPheGlyIleIleTrpLeu                                 140145150                                                                     (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 amino acids                                                    (B) TYPE: amino acid                                                         (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       ArgLysArgLysProValAlaHisValValAlaAsnProGl nAla                                151015                                                                        GluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeuLeu                                 202530                                                                        AlaAsn GlyValGluLeuArgAspAsnGlnLeuValValProSer                                354045                                                                        GluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGlyGln                                 50 5560                                                                       GlyCysProSerThrHisValLeuLeuThrHisThrIleSerArg                                 657075                                                                        IleAlaValSerTyrGlnThrLysValAs nLeuLeuSerAlaIle                                808590                                                                        LysSerProCysGlnArgGluThrProGluGlyAlaGluAlaLys                                 95100 105                                                                     ProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeuGlu                                 110115120                                                                     LysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyrLeu                                  125130135                                                                    AspPheAlaGluSerGlyGlnValTyrPheGlyIleIlePheLeu                                 140145150                                                                     (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 150 amino acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      ArgLysArgLysProValAlaHisV alValAlaAsnProGlnAla                                151015                                                                        GluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeuLeu                                 2025 30                                                                       AlaAsnGlyValGluLeuArgAspAsnGlnLeuValValProSer                                 354045                                                                        GluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGlyGln                                  505560                                                                       GlyCysProSerThrHisValLeuLeuThrHisThrIleSerArg                                 657075                                                                        IleAlaValSerTy rGlnThrLysValAsnLeuLeuSerAlaIle                                808590                                                                        LysSerProCysGlnArgGluThrProGluGlyAlaGluAlaLys                                 95 100105                                                                     ProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeuGlu                                 110115120                                                                     LysGlyAspArgLeuSerAlaGluIleAsnArgPro AspTyrLeu                                125130135                                                                     AspPheAlaGluSerGlyGlnValTyrPheGlyIleIlePhePhe                                 140145150                                                                     ( 2) INFORMATION FOR SEQ ID NO:11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      ArgLysArg LysProValAlaHisValValAlaAsnProGlnAla                                151015                                                                        GluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeuLeu                                 20 2530                                                                       AlaAsnGlyValGluLeuArgAspAsnGlnLeuValValProSer                                 354045                                                                        GluGlyLeuTyrLeuIleTyrSerGlnValLeu PheLysGlyGln                                505560                                                                        GlyCysProSerThrHisValLeuLeuThrHisThrIleSerArg                                 65707 5                                                                       IleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAlaIle                                 808590                                                                        LysSerProCysGlnArgGluThrProGluGlyAlaGluAlaLys                                  95100105                                                                     ProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeuGlu                                 110115120                                                                     LysGlyAspArgLeuSerAla GluIleAsnArgProAspTyrLeu                                125130135                                                                     AspPheAlaGluSerGlyGlnValTyrPheGlyIleIleTrpPhe                                 140145 150                                                                    (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi ) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                     ArgLysArgLysProValAlaHisValValAlaAsnProGlnAla                                 151015                                                                        GluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeuLeu                                  202530                                                                       AlaAsnGlyValGluLeuArgAspAsnGlnLeuValValProSer                                 354045                                                                        GluGlyLeuTyrLeuI leTyrSerGlnValLeuPheLysGlyGln                                505560                                                                        GlyCysProSerThrHisValLeuLeuThrHisThrIleSerArg                                 65 7075                                                                       IleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAlaIle                                 808590                                                                        LysSerProCysGlnArgGluThrProGluGlyAlaGlu AlaLys                                95100105                                                                      ProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeuGlu                                 110115120                                                                     LysGl yAspArgLeuSerAlaGluIleAsnArgProAspTyrLeu                                125130135                                                                     AspPheAlaGluSerGlyGlnValTyrPheGlyIlePheAlaLeu                                 140 145150                                                                    (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ArgLysArgLysProValAlaHisValValAlaAsnProGlnAla                                 151015                                                                        GluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsn AlaLeuLeu                                202530                                                                        AlaAsnGlyValGluLeuArgAspAsnGlnLeuValValProSer                                 354045                                                                        G luGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGlyGln                                505560                                                                        GlyCysProSerThrHisValLeuLeuThrHisThrIleSerArg                                  657075                                                                       IleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAlaIle                                 808590                                                                        LysSerProCysGlnArgGluThr ProGluGlyAlaGluAlaLys                                95100105                                                                      ProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeuGlu                                 110115 120                                                                    LysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyrLeu                                 125130135                                                                     AspPheAlaGluSerGlyGlnValTyrPheGlyPheIleAlaLeu                                  140145150                                                                    (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                   (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ArgLysArgLysProValAlaHisValValAlaAsnProGlnAla                                 151015                                                                        GluGlyGlnLeuGlnTrpLe uAsnArgArgAlaAsnAlaLeuLeu                                202530                                                                        AlaAsnGlyValGluLeuArgAspAsnGlnLeuValValProSer                                 3540 45                                                                       GluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGlyGln                                 505560                                                                        GlyCysProSerThrHisValLeuLeuThrHisThrIleSer Arg                                657075                                                                        IleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAlaIle                                 808590                                                                        LysSerPr oCysGlnArgGluThrProGluGlyAlaGluAlaLys                                95100105                                                                      ProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeuGlu                                 110 115120                                                                    LysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyrLeu                                 125130135                                                                     AspPheAlaGluSerGlyGlnValTyrPheG lyIleIleAlaLeu                                140145150                                                                     (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 148 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 ( A) NAME/KEY:                                                                (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      MetLysProValAlaHisValValAlaAsnProGlnAlaGluGly                                 151015                                                                        GlnL euGlnTrpLeuAsnArgArgAlaAsnAlaLeuLeuAlaAsn                                202530                                                                        GlyValGluLeuArgAspAsnGlnLeuValValProSerGluGly                                 35 4045                                                                       LeuTyrLeuIleTyrSerGlnValLeuPheLysGlyGlnGlyCys                                 505560                                                                        ProSerThrHisValLeuLeuThrHis ThrIleSerArgIleAla                                657075                                                                        ValSerTyrGlnThrLysValAsnLeuLeuSerAlaIleLysSer                                 8085 90                                                                       ProCysGlnArgGluThrProGluGlyAlaGluAlaLysProTrp                                 95100105                                                                      TyrGluProIleTyrLeuGlyGlyValPheGlnLeuGluLysGly                                  110115120                                                                    AspArgLeuSerAlaGluIleAsnArgProAspTyrLeuAspPhe                                 125130135                                                                     AlaGluSerGlyGln ValTyrPheGlyIleIleAlaPhe                                      140145                                                                        (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 149 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                  (B) LOCATION:                                                                (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      MetAspLysProValAlaHisValValAlaAsnProGlnAlaGlu                                 151015                                                                        GlyGlnLeu GlnTrpLeuAsnArgArgAlaAsnAlaLeuLeuAla                                202530                                                                        AsnGlyValGluLeuArgAspAsnGlnLeuValValProSerGlu                                 35 4045                                                                       GlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGlyGlnGly                                 505560                                                                        CysProSerThrHisValLeuLeuThrHisTh rIleSerArgIle                                657075                                                                        AlaValSerTyrGlnThrLysValAsnLeuLeuSerAlaIleLys                                 80859 0                                                                       SerProCysGlnArgGluThrProGluGlyAlaGluAlaLysPro                                 95100105                                                                      TrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeuGluLys                                  110115120                                                                    GlyAspArgLeuSerAlaGluIleAsnArgProAspTyrLeuAsp                                 125130135                                                                     PheAlaGluSerGlyGlnVal TyrPheGlyIleIleAlaPhe                                   140145                                                                        (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                  (B) LOCATION:                                                                (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      MetSerAspLysProValAlaHisValValAlaAsnProGlnAla                                 151015                                                                        GluGlyGlnLe uGlnTrpLeuAsnArgArgAlaAsnAlaLeuLeu                                202530                                                                        AlaAsnGlyValGluLeuArgAspAsnGlnLeuValValProSer                                 35 4045                                                                       GluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGlyGln                                 505560                                                                        GlyCysProSerThrHisValLeuLeuThrHisT hrIleSerArg                                657075                                                                        IleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAlaIle                                 808590                                                                         LysSerProCysGlnArgGluThrProGluGlyAlaGluAlaLys                                95100105                                                                      ProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeuGlu                                  110115120                                                                    LysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyrLeu                                 125130135                                                                     AspPheAlaGluSerGlyGlnVa lTyrPheGlyIleIleAlaPhe                                140145150                                                                     (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 151 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      MetProSerAspLysProValAlaHisValValAlaAsnProGln                                 1510 15                                                                       AlaGluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeu                                 202530                                                                        LeuAlaAsnGlyValGluLeuArgAspAsnGlnLeuValValPro                                  354045                                                                       SerGluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGly                                 505560                                                                        GlnGlyCysProSerThrH isValLeuLeuThrHisThrIleSer                                657075                                                                        ArgIleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAla                                 8085 90                                                                       IleLysSerProCysGlnArgGluThrProGluGlyAlaGluAla                                 95100105                                                                      LysProTrpTyrGluProIleTyrLeuGlyGlyValPheGln Leu                                110115120                                                                     GluLysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyr                                 125130135                                                                     LeuAspP heAlaGluSerGlyGlnValTyrPheGlyIleIleAla                                140145150                                                                     Phe                                                                           (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 151 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      MetArgLysArgLysProValAlaHisValValAlaAsnProGln                                 15 1015                                                                       AlaGluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeu                                 202530                                                                        LeuAlaAsnGlyValGluLeuArgAspAsnGln LeuValValPro                                354045                                                                        SerGluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGly                                 505560                                                                        GlnGlyCysProSerThrHisValLeuLeuThrHisThrIleSer                                 657075                                                                        ArgIleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAla                                  808590                                                                       IleLysSerProCysGlnArgGluThrProGluGlyAlaGluAla                                 95100105                                                                      LysProTrpTyrGluProIle TyrLeuGlyGlyValPheGlnLeu                                110115120                                                                     GluLysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyr                                 125130 135                                                                    LeuAspPheAlaGluSerGlyGlnValTyrPheGlyIleIleAla                                 140145150                                                                     Phe                                                                           (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 151 amino acids                                                    (B) TYPE: amino acid                                                         (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      MetArgLysArgLysProValAlaHisValValAlaAsnProGln                                 1 51015                                                                       AlaGluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeu                                 202530                                                                        LeuAlaAsnGlyV alGluLeuArgAspAsnGlnLeuValValPro                                354045                                                                        SerGluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGly                                 50 5560                                                                       GlnGlyCysProSerThrHisValLeuLeuThrHisThrIleSer                                 657075                                                                        ArgIleAlaValSerTyrGlnThrLysValAsnLeu LeuSerAla                                808590                                                                        IleLysSerProCysGlnArgGluThrProGluGlyAlaGluAla                                 95100105                                                                      L ysProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeu                                110115120                                                                     GluLysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyr                                 1 25130135                                                                    LeuAspPheAlaGluSerGlyGlnValTyrPheGlyIleIleAla                                 140145150                                                                     Trp                                                                           (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 152 amino acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      MetArgLysArgLysProValAlaHis ValValAlaAsnProGln                                151015                                                                        AlaGluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeu                                 2025 30                                                                       LeuAlaAsnGlyValGluLeuArgAspAsnGlnLeuValValPro                                 354045                                                                        SerGluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGly                                  505560                                                                       GlnGlyCysProSerThrHisValLeuLeuThrHisThrIleSer                                 657075                                                                        ArgIleAlaValSer TyrGlnThrLysValAsnLeuLeuSerAla                                808590                                                                        IleLysSerProCysGlnArgGluThrProGluGlyAlaGluAla                                 95 100105                                                                     LysProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeu                                 110115120                                                                     GluLysGlyAspArgLeuSerAlaGluIleAsnArgPro AspTyr                                125130135                                                                     LeuAspPheAlaGluSerGlyGlnValTyrPheGlyIleIleAla                                 140145150                                                                     LeuP he                                                                       (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 151 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      MetA rgLysArgLysProValAlaHisValValAlaAsnProGln                                151015                                                                        AlaGluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeu                                 20 2530                                                                       LeuAlaAsnGlyValGluLeuArgAspAsnGlnLeuValValPro                                 354045                                                                        SerGluGlyLeuTyrLeuIleTyrSer GlnValLeuPheLysGly                                505560                                                                        GlnGlyCysProSerThrHisValLeuLeuThrHisThrIleSer                                 6570 75                                                                       ArgIleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAla                                 808590                                                                        IleLysSerProCysGlnArgGluThrProGluGlyAlaGluAla                                  95100105                                                                     LysProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeu                                 110115120                                                                     GluLysGlyAspArg LeuSerAlaGluIleAsnArgProAspTyr                                125130135                                                                     LeuAspPheAlaGluSerGlyGlnValTyrPheGlyIleIleTrp                                 140 145150                                                                    Leu                                                                           (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 151 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D ) OTHER INFORMATION:                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      MetArgLysArgLysProValAlaHisValValAlaAsnProGln                                 151015                                                                        AlaGluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAl aLeu                                202530                                                                        LeuAlaAsnGlyValGluLeuArgAspAsnGlnLeuValValPro                                 354045                                                                        SerGlu GlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGly                                505560                                                                        GlnGlyCysProSerThrHisValLeuLeuThrHisThrIleSer                                 65 7075                                                                       ArgIleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAla                                 808590                                                                        IleLysSerProCysGlnArgGluThrPro GluGlyAlaGluAla                                95100105                                                                      LysProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeu                                 110115 120                                                                    GluLysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyr                                 125130135                                                                     LeuAspPheAlaGluSerGlyGlnValTyrPheGlyIleIlePhe                                  140145150                                                                    Leu                                                                           (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 151 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                   (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      MetArgLysArgLysProValAlaHisValValAlaAsnProGln                                 151015                                                                        AlaGluGlyGlnLeuGlnTrp LeuAsnArgArgAlaAsnAlaLeu                                202530                                                                        LeuAlaAsnGlyValGluLeuArgAspAsnGlnLeuValValPro                                 3540 45                                                                       SerGluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGly                                 505560                                                                        GlnGlyCysProSerThrHisValLeuLeuThrHisThrIleSer                                 657075                                                                        ArgIleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAla                                 808590                                                                        IleLysSer ProCysGlnArgGluThrProGluGlyAlaGluAla                                95100105                                                                      LysProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeu                                 110 115120                                                                    GluLysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyr                                 125130135                                                                     LeuAspPheAlaGluSerGlyGlnValTyrPhe GlyIleIlePhe                                140145150                                                                     Phe                                                                           (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 151 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      MetArgLysArgLysProValAlaHisValValAlaAsnProGln                                 151015                                                                         AlaGluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeu                                202530                                                                        LeuAlaAsnGlyValGluLeuArgAspAsnGlnLeuValValPro                                  354045                                                                       SerGluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGly                                 505560                                                                        GlnGlyCysProSerThrHisVal LeuLeuThrHisThrIleSer                                657075                                                                        ArgIleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAla                                 8085 90                                                                       IleLysSerProCysGlnArgGluThrProGluGlyAlaGluAla                                 95100105                                                                      LysProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeu                                  110115120                                                                    GluLysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyr                                 125130135                                                                     LeuAspPheAla GluSerGlyGlnValTyrPheGlyIleIleTrp                                140145150                                                                     Phe                                                                           (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 151 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      MetArgLysArgLysProValAlaHisValValAlaAsnProGln                                 15 1015                                                                       AlaGluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeu                                 202530                                                                        LeuAlaAsnGlyValGluLeuArgAspAsnGlnLeuVal ValPro                                354045                                                                        SerGluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGly                                 505560                                                                        GlnG lyCysProSerThrHisValLeuLeuThrHisThrIleSer                                657075                                                                        ArgIleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAla                                 80 8590                                                                       IleLysSerProCysGlnArgGluThrProGluGlyAlaGluAla                                 95100105                                                                      LysProTrpTyrGluProIleTyrLeu GlyGlyValPheGlnLeu                                110115120                                                                     GluLysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyr                                 125130 135                                                                    LeuAspPheAlaGluSerGlyGlnValTyrPheGlyIlePheAla                                 140145150                                                                     Leu                                                                           (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 151 amino acids                                                   (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      MetArgLysArgLysProValAlaHisValValAlaAsnProGln                                 1 51015                                                                       AlaGluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeu                                 202530                                                                        LeuAlaAsnGlyValGlu LeuArgAspAsnGlnLeuValValPro                                354045                                                                        SerGluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGly                                 5055 60                                                                       GlnGlyCysProSerThrHisValLeuLeuThrHisThrIleSer                                 657075                                                                        ArgIleAlaValSerTyrGlnThrLysValAsnLeuLeuSe rAla                                808590                                                                        IleLysSerProCysGlnArgGluThrProGluGlyAlaGluAla                                 95100105                                                                      LysPro TrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeu                                110115120                                                                     GluLysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyr                                 125 130135                                                                    LeuAspPheAlaGluSerGlyGlnValTyrPheGlyPheIleAla                                 140145150                                                                     Leu                                                                           (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 151 amino acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      MetArgLysArgLysProValAlaHisValVal AlaAsnProGln                                151015                                                                        AlaGluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeu                                 202530                                                                        LeuAlaAsnGlyValGluLeuArgAspAsnGlnLeuValValPro                                 354045                                                                        SerGluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGly                                  505560                                                                       GlnGlyCysProSerThrHisValLeuLeuThrHisThrIleSer                                 657075                                                                        ArgIleAlaValSerTyrGln ThrLysValAsnLeuLeuSerAla                                808590                                                                        IleLysSerProCysGlnArgGluThrProGluGlyAlaGluAla                                 95100 105                                                                     LysProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeu                                 110115120                                                                     GluLysGlyAspArgLeuSerAlaGluIleAsnArgProAspTy r                                125130135                                                                     LeuAspPheAlaGluSerGlyGlnValTyrPheGlyIleIleAla                                 140145150                                                                     Leu                                                                           (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      ProSerAspLys ProValAlaHisValValAlaAsnProGlnAla                                151015                                                                        GluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeuLeu                                 20 2530                                                                       AlaAsnGlyValGluLeuArgAspAsnGlnLeuValValProSer                                 354045                                                                        GluAspLeuTyrLeuIleTyrSerGlnValLeuPh eLysGlyGln                                505560                                                                        GlyCysProSerThrHisValLeuLeuThrHisThrIleSerArg                                 657075                                                                         IleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAlaIle                                808590                                                                        LysSerProCysGlnArgGluThrProGluGlyAlaGluAlaLys                                  95100105                                                                     ProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeuGlu                                 110115120                                                                     LysGlyAspArgLeuSerAlaGlu IleAsnArgProAspTyrLeu                                125130135                                                                     AspPheAlaGluSerGlyGlnValTyrPheGlyIleIleAlaLeu                                 140145 150                                                                    (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 151 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      MetProSerAspLysProValAlaHisValValAlaAsnProGln                                 151015                                                                        AlaGluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeu                                  202530                                                                       LeuAlaAsnGlyValGluLeuArgAspAsnGlnLeuValValPro                                 354045                                                                        SerGluAspLeuTyrLeuIl eTyrSerGlnValLeuPheLysGly                                505560                                                                        GlnGlyCysProSerThrHisValLeuLeuThrHisThrIleSer                                 6570 75                                                                       ArgIleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAla                                 808590                                                                        IleLysSerProCysGlnArgGluThrProGluGlyAlaGluA la                                95100105                                                                      LysProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeu                                 110115120                                                                     GluLysGl yAspArgLeuSerAlaGluIleAsnArgProAspTyr                                125130135                                                                     LeuAspPheAlaGluSerGlyGlnValTyrPheGlyIleIleAla                                 140 145150                                                                    Leu                                                                           (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      ProSerAspLysProValAlaHisValValAlaAsnProGlnAla                                 151015                                                                        GluGlyGlnLeuGlnTrpLeuAsnArgArgAlaA snAlaLeuLeu                                202530                                                                        AlaAsnGlyValGluLeuArgAspAsnGlnLeuValValProSer                                 354045                                                                         GluAspLeuTyrLeuIleTyrSerGlnValLeuPheLysGlyGln                                505560                                                                        GlyCysProSerThrHisValLeuLeuThrHisThrIleSerArg                                  657075                                                                       IleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAlaIle                                 808590                                                                        LysSerProCysGlnArgGluT hrProGluGlyAlaGluAlaLys                                95100105                                                                      ProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeuGlu                                 110115 120                                                                    LysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyrLeu                                 125130135                                                                     AspPheAlaGluSerGlyGlnValTyrPheGlyIleIleAlaPhe                                  140145150                                                                    (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 151 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      MetProSerAspLysProValAlaHisValValAlaAsnProGln                                 151015                                                                        AlaGluGlyGlnLeuGln TrpLeuAsnArgArgAlaAsnAlaLeu                                202530                                                                        LeuAlaAsnGlyValGluLeuArgAspAsnGlnLeuValValPro                                 3540 45                                                                       SerGluAspLeuTyrLeuIleTyrSerGlnValLeuPheLysGly                                 505560                                                                        GlnGlyCysProSerThrHisValLeuLeuThrHisThrIle Ser                                657075                                                                        ArgIleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAla                                 808590                                                                        IleLysS erProCysGlnArgGluThrProGluGlyAlaGluAla                                95100105                                                                      LysProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeu                                 110 115120                                                                    GluLysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyr                                 125130135                                                                     LeuAspPheAlaGluSerGlyGlnValTyr PheGlyIleIleAla                                140145150                                                                     Phe                                                                           (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 157 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      ValArgSerSerSerArgThrProSerAspLysProValAlaHis                                 151015                                                                        ValValAlaAsnProGlnAlaGluGlyGlnLeuGlnTrpLeuAsn                                 202530                                                                        ArgArgAlaAsnAlaLeuLeuAlaAsnGlyValGluLeuArgAsn                                  354045                                                                       AsnSerLeuValValProSerGluGlyLeuTyrLeuIleTyrSer                                 505560                                                                        GlnValLeuPheLysGlyGln GlyCysProSerThrHisValLeu                                657075                                                                        LeuThrHisThrIleSerArgIleAlaValSerTyrGlnThrLys                                 8085 90                                                                       ValAsnLeuLeuSerAlaIleLysSerProCysGlnArgGluThr                                 95100105                                                                      ProGluGlyAlaGluAlaLysProTrpTyrGluProIleTyrLeu                                 110115120                                                                     GlyGlyValPheGlnLeuGluLysGlyAspArgLeuSerAlaGlu                                 125130135                                                                     IleAsnArgP roAspTyrLeuAspPheAlaGluSerGlyGlnVal                                140145150                                                                     TyrPheGlyIleIleAlaLeu                                                         155                                                                           (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 158 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      MetValArgSerSerSerArgThrProSerAspLysP roValAla                                151015                                                                        HisValValAlaAsnProGlnAlaGluGlyGlnLeuGlnTrpLeu                                 202530                                                                        Asn ArgArgAlaAsnAlaLeuLeuAlaAsnGlyValGluLeuArg                                354045                                                                        AsnAsnSerLeuValValProSerGluGlyLeuTyrLeuIleTyr                                 50 5560                                                                       SerGlnValLeuPheLysGlyGlnGlyCysProSerThrHisVal                                 657075                                                                        LeuLeuThrHisThrIleSerArgIle AlaValSerTyrGlnThr                                808590                                                                        LysValAsnLeuLeuSerAlaIleLysSerProCysGlnArgGlu                                 95100 105                                                                     ThrProGluGlyAlaGluAlaLysProTrpTyrGluProIleTyr                                 110115120                                                                     LeuGlyGlyValPheGlnLeuGluLysGlyAspArgLeuSerAla                                  125130135                                                                    GluIleAsnArgProAspTyrLeuAspPheAlaGluSerGlyGln                                 140145150                                                                     ValTyrPheGlyIle IleAlaLeu                                                     155                                                                           (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      ProSerAspLysProValAlaHisValValAlaAsnProGlnAla                                 151015                                                                        GluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAla LeuLeu                                202530                                                                        AlaAsnGlyValGluLeuArgAsnAsnSerLeuValValProSer                                 354045                                                                        GluG lyLeuTyrLeuIleTyrSerGlnValLeuPheLysGlyGln                                505560                                                                        GlyCysProSerThrHisValLeuLeuThrHisThrIleSerArg                                 65 7075                                                                       IleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAlaIle                                 808590                                                                        LysSerProCysGlnArgGluThrPro GluGlyAlaGluAlaLys                                95100105                                                                      ProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeuGlu                                 110115 120                                                                    LysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyrLeu                                 125130135                                                                     AspPheAlaGluSerGlyGlnValTyrPheGlyIleIleAlaLeu                                  140145150                                                                    (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 151 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                   (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      MetProSerAspLysProValAlaHisValValAlaAsnProGln                                 151015                                                                        AlaGluGlyGlnLeuGlnTrpL euAsnArgArgAlaAsnAlaLeu                                202530                                                                        LeuAlaAsnGlyValGluLeuArgAsnAsnSerLeuValValPro                                 3540 45                                                                       SerGluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGly                                 505560                                                                        GlnGlyCysProSerThrHisValLeuLeuThrHisThrIleSer                                  657075                                                                       ArgIleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAla                                 808590                                                                        IleLysSerPr oCysGlnArgGluThrProGluGlyAlaGluAla                                95100105                                                                      LysProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeu                                 110 115120                                                                    GluLysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyr                                 125130135                                                                     LeuAspPheAlaGluSerGlyGlnValTyrPhe GlyIleIleAla                                140145150                                                                     Leu                                                                           (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      ProSerAspLysProValAlaHisValValAlaAsnProGlnAla                                 151015                                                                        Gl uGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeuLeu                                202530                                                                        AlaAsnGlyValGluLeuArgAsnAsnSerLeuValValProSer                                 3 54045                                                                       GluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGlyGln                                 505560                                                                        GlyCysProSerThrHisValLeu LeuThrHisThrIleSerArg                                657075                                                                        IleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAlaIle                                 8085 90                                                                       LysSerProCysGlnArgGluThrProGluGlyAlaGluAlaLys                                 95100105                                                                      ProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeuGlu                                  110115120                                                                    LysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyrLeu                                 125130135                                                                     AspPheAlaGluS erGlyGlnValTyrPheGlyIleIleAlaPhe                                140145150                                                                     (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 151 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      MetProSerAspLysProValAlaHisValValAlaAsnProGln                                 1510 15                                                                       AlaGluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeu                                 202530                                                                        LeuAlaAsnGlyValGluLeuArgAsnAsnSerLeuValValP ro                                354045                                                                        SerGluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGly                                 505560                                                                        GlnGlyCys ProSerThrHisValLeuLeuThrHisThrIleSer                                657075                                                                        ArgIleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAla                                 80 8590                                                                       IleLysSerProCysGlnArgGluThrProGluGlyAlaGluAla                                 95100105                                                                      LysProTrpTyrGluProIleTyrLeuGlyGl yValPheGlnLeu                                110115120                                                                     GluLysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyr                                 12513013 5                                                                    LeuAspPheAlaGluSerGlyGlnValTyrPheGlyIleIleAla                                 140145150                                                                     Phe                                                                           (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                     (D) TOPOLOGY: circular                                                        (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      ATTATTGCCTTC12                                                                (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      ATTATTGCCTGG12                                                                (2) INFORMATION FOR SEQ ID NO:41:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      ATTATTGCCCTGTTC 15                                                            (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      ATTATTTGGCTG12                                                                (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      ATTATTTTCCTG12                                                                (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      ATTATTTTCTTC12                                                                (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      ATTATTTGGTTC12                                                                (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: circular                                                       (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      ATTTTCGCCCTG12                                                                (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                     (D) TOPOLOGY: circular                                                        (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      TTCATTGCCCTG12                                                                (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 429 base pairs                                                     (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      AAGCCTGTAGCCCATGTTGTAGCAAACCCTCAAGCTGAGGGGCAGCTCCA 50                         GTGGCTGAACCGCCGGGCCAATGCCCTGCTGGCCAATGGCGTGGAGCTGA100                         GAGATAACCAGCTGGTGGTACCATCAGAGGGCCTGTACCTCATCTACTCC150                         CAGGTCCTCTTCAAGGGCCAAGGCTGCCCGTCGACCCATGTGCTCCTCAC200                         CCACACCAT CAGCCGCATCGCCGTCTCCTACCAGACCAAGGTCAACCTCC250                        TCTCTGCGATCAAGAGCCCCTGCCAGAGGGAGACCCCAGAGGGGGCTGAG300                         GCCAAGCCATGGTATGAGCCCATCTATCTGGGAGGGGTCTTCCAGCTGGA350                         GAAGGGTGACCGACTCAGCGC TGAAATCAATCGGCCCGACTATCTCGACT400                        TTGCCGAGTCTGGGCAGGTCTACTTTGGG429                                              (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 103 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                         (ix) FEATURE:                                                                (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      CATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAAT50                          CATCGAACTAGTTAACTAGTACGCAAGTTCACGTAAAAAGGGTA TCGATA100                        ATG103                                                                        (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                  (B) LOCATION:                                                                (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      TGATAAGCTTAGCCCGCCTAATGAGCGGGCTTTTTTTT38                                  

What we claim is:
 1. A modified TNF polypeptide represented by formula (I)

    (NH.sub.2)-(Met).sub.n -A-X-B-(COOH)                       (I)

wherein n is 0 or 1, A denotes -Arg-Lys-Arg-, B denotes -Ile-Ile-Ala-Phe-, -Ile-Ile-Ala-Trp-, -Ile-Ile-Trp-Leu, or Ile-Ile-Phe-Leu-, X denotes -Lys-Pro-Val-Ala-His-Val-Val-Ala-Asn-Pro-Gln-Ala-Glu-Gly-Gln-Leu-Gln-Trp-Leu-Asn-Arg-Arg-Ala-Asn-Ala-Leu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-Asp-Asn-Gln-Leu-Val-Val-Pro-Ser-Glu-Gly-Leu-Tyr-Leu-Ile-Tyr-Ser-Gln-Val-Leu-Phe-Lys-Gly-Gln-Gly-Cys-Pro-Ser-Thr-His-Val-Leu-Leu-Thr-His-Thr-Ile-Ser-Arg-Ile-Ala-Val-Ser-Tyr-Gln-Thr-Lys-Val-Asn-Leu-Leu-Ser-Ala-Ile-Lys-Ser-Pro-Cys-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Tyr-Glu-Pro-Ile-Tyr-Leu-Gly-Gly-Val-Phe-Gln-Leu-Glu-Lys-Gly-Asp-Arg-Leu-Ser-Ala-Glu-Ile-Asn-Arg-Pro-Asp-Typ-Leu-Asp-Phe-Ala-Glu-Ser-Gly-Gln-Val-Tyr-Phe-Gly-.
 2. A recombinant plasmic comprising a DNA sequence encoding the polypeptide of formula (I)

    (NH.sub.2)-(Met).sub.n -A-X-B-(COOH)                       (I)

wherein n is 0 or 1, A denotes -Arg-Lys-Arg-, B denotes -Ile-Ile-Ala-Phe-, -Ile-Ile-Ala-Trp-, -Ile-Ile-Trp-Leu, or Ile-Ile-Phe-Leu-, X denotes -Lys-Pro-Val-Ala-His-Val-Val-Ala-Asn-Pro-Gln-Ala-Glu-Gly-Gln-Leu-Gln-Trp-Leu-Asn-Arg-Arg-Ala-Asn-Ala-Leu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-Asp-Asn-Gln-Leu-Val-Val-Pro-Ser-Glu-Gly-Leu-Tyr-Leu-Ile-Tyr-Ser-Gln-Val-Leu-Phe-Lys-Gly-Gln-Gly-Cys-Pro-Ser-Thr-His-Val-Leu-Leu-Thr-His-Thr-Ile-Ser-Arg-Ile-Ala-Val-Ser-Tyr-Gln-Thr-Lys-Val-Asn-Leu-Leu-Ser-Ala-Ile-Lys-Ser-Pro-Cys-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Tyr-Glu-Pro-Ile-Tyr-Leu-Gly-Gly-Val-Phe-Gln-Leu-Glu-Lys-Gly-Asp-Arg-Leu-Ser-Ala-Glu-Ile-Asn-Arg-Pro-Asp-Tyr-Leu-Asp-Phe-Ala-Glu-Ser-Gly-Gln-Val-Tyr-Phe-Gly-.
 3. The plasmid of claim 1 which is pTNF616, pTNF617, pTNF618 or pTNF619.
 4. The plasmid of claim 1 which is transformed with the recombinant plasmid having the DNA sequence encoding the modified TNF polypeptide of formula (I)

    (NH.sub.2)-(Met).sub.n -A-X-B-(COOH)                       (I)

wherein n is 0 or 1, A denotes -Arg-Lys-Arg-, B denotes -Ile-Ile-Ala-Phe-, -Ile-Ile-Ala-Trp-, -Ile-Ile-Trp-Leu, or Ile-Ile-Phe-Leu-, X denotes -Lys-Pro-val-Ala-His-Val-Val-Ala-Asn-Pro-Gln-Ala-Glu-Gly-Gln-Leu-Gln-Trp-Leu-Asn-Arg-Arg-Ala-Asn-Ala-Leu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-Asp-Asn-Gln-Leu-Val-Val-Pro-Ser-Glu-Gly-Leu-Tyr-Leu-Ile-Tyr-Ser-Gln-Val-Leu-Phe-Lys-Gly-Gln-Gly-Cys-Pro-Ser-Thr-His-Val-Leu-Leu-Thr-His-Thr-Ile-Ser-Arg-Ile-Ala-Val-Ser-Tyr-Gln-Thr-Lys-Val-Asn-Leu-Leu-Ser-Ala-Ile-Lys-Ser-Pro-Cys-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Tyr-Glu-Pro-Ile-Tyr-Leu-Gly-Gly-Val-Phe-Gln-Leu-Glu-Lys-Gly-Asp-Arg-Leu-Ser-Ala-Glu-Ile-Asn-Arg-Pro-Asp-Tyr-Leu-Asp-Phe-Ala-Glu-Ser-Gly-Gln-Val-Tyr-Phe-Gly-.
 5. The unicellular microorganism of claim 4 which is Escherichia coli.
 6. A process for producing the modified TNF polypeptide of formula (I)

    (NH.sub.2)-(Met).sub.n -A-X-B-(COOH)                       (I)

wherein n is 0 or 1, A denotes -Arg-Lys-Arg-, B denotes -Ile-Ile-Ala-Phe-, -Ile-Ile-Ala-Trp-, -Ile-Ile-Trp-Leu, or Ile-Ile-Phe-Leu-, X denotes -Lys-Pro-Val-Ala-His-Val-Val-Ala-Asn-Pro-Gln-Ala-Glu-Gly-Gln-Leu-Gln-Trp-Leu-Asn-Arg-Arg-Ala-Asn-Ala-Leu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-Asp-Asn-Gln-Leu-Val-Val-Pro-Ser-Glu-Gly-Leu-Tyr-Leu-Ile-Tyr-Ser-Gln-Val-Leu-Phe-Lys-Gly-Gln-Gly-Cys-Pro-Ser-Thr-His-Val-Leu-Leu-Thr-His-Thr-Ile-Ser-Arg-Ile-Ala-Val-Ser-Tyr-Gln-Thr-Lys-Val-Asn-Leu-Leu-Ser-Ala-Ile-Lys-Ser-Pro-Cys-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Tyr-Glu-Pro-Ile-Tyr-Leu-Gly-Gly-Val-Phe-Gln-Leu-Glu-Lys-Gly-Asp-Arg-Leu-Ser-Ala-Glu-Ile-Asn-Arg-Pro-Asp-Typ-Leu-Asp-Phe-Ala-Glu-Ser-Gly-Gln-Val-Tyr-Phe-Gly-,which comprises cultivating an Escherichia coli which is transformed with a DNA sequence encoding the polypeptide of formula (I) to produce and accumulate the polypeptide of formula (I) in a culture broth, and separating the polypeptide from the resulting culture broth.
 7. A pharmaceutical composition comprising an antitumorally effective amount of the modified TNF polypeptide of formula (I)

    (NH.sub.2)-(Met).sub.n -A-X-B-(COOH)                       (I)

wherein n is 0 or 1, A denotes -Arg-Lys-Arg-, B denotes -Ile-Ile-Ala-Phe-, -Ile-Ile-Ala-Trp-, -Ile-Ile-Trp-Leu, or Ile-Ile-Phe-Leu-, X denotes -Lys-Pro-Val-Ala-His-Val-Val-Ala-Asn-Pro-Gln-Ala-Glu-Gly-Gln-Leu-Gln-Trp-Leu-Asn-Arg-Arg-Ala-Asn-Ala-Leu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-Asp-Asn-Gln-Leu-Val-Val-Pro-Ser-Glu-Gly-Leu-Tyr-Leu-Ile-Tyr-Ser-Gln-Val-Leu-Phe-Lys-Gly-Gln-Gly-Cys-Pro-Ser-Thr-His-Val-Leu-Leu-Thr-His-Thr-Ile-Ser-Arg-Ile-Ala-Val-Ser-Tyr-Gln-Thr-Lys-Val-Asn-Leu-Leu-Ser-Ala-Ile-Lys-Ser-Pro-Cys-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Tyr-Glu-Pro-Ile-Tyr-Leu-Gly-Gly-Val-Phe-Gln-Leu-Glu-Lys-Gly-Asp-Arg-Leu-Ser-Ala-Glu-Ile-Asn-Arg-Pro-Asp-Tyr-Leu-Asp-Phe-Ala-Glu-Ser-Gly-Gln-Val-Tyr-Phe-Gly-,and a pharmaceutically acceptable amount of a carrier. 